The Interdependency and Co-Regulation of the Vitamin D and Cholesterol Metabolism.

Vitamin D and cholesterol metabolism overlap significantly in the pathways that contribute to their biosynthesis. However, our understanding of their independent and co-regulation is limited. Cardiovascular disease is the leading cause of death globally and atherosclerosis, the pathology associated with elevated cholesterol, is the leading cause of cardiovascular disease. It is therefore important to understand vitamin D metabolism as a contributory factor. From the literature, we compile evidence of how these systems interact, relating the understanding of the molecular mechanisms involved to the results from observational studies. We also present the first systems biology pathway map of the joint cholesterol and vitamin D metabolisms made available using the Systems Biology Graphical Notation (SBGN) Markup Language (SBGNML). It is shown that the relationship between vitamin D supplementation, total cholesterol, and LDL-C status, and between latitude, vitamin D, and cholesterol status are consistent with our knowledge of molecular mechanisms. We also highlight the results that cannot be explained with our current knowledge of molecular mechanisms: (i) vitamin D supplementation mitigates the side-effects of statin therapy; (ii) statin therapy does not impact upon vitamin D status; and critically (iii) vitamin D supplementation does not improve cardiovascular outcomes, despite improving cardiovascular risk factors. For (iii), we present a hypothesis, based on observations in the literature, that describes how vitamin D regulates the balance between cellular and plasma cholesterol. Answering these questions will create significant opportunities for advancement in our understanding of cardiovascular health.

In vitro and in silico multidimensional modeling of oncolytic tumor virotherapy dynamics.

Tumor therapy with replication competent viruses is an exciting approach to cancer eradication where viruses are engineered to specifically infect, replicate, spread and kill tumor cells. The outcome of tumor virotherapy is complex due to the variable interactions between the cancer cell and virus populations as well as the immune response. Oncolytic viruses are highly efficient in killing tumor cells in vitro, especially in a 2D monolayer of tumor cells, their efficiency is significantly lower in a 3D environment, both in vitro and in vivo. This indicates that the spatial dimension may have a major influence on the dynamics of virus spread. We study the dynamic behavior of a spatially explicit computational model of tumor and virus interactions using a combination of in vitro 2D and 3D experimental studies to inform the models. We determine the number of nearest neighbor tumor cells in 2D (median = 6) and 3D tumor spheroids (median = 16) and how this influences virus spread and the outcome of therapy. The parameter range leading to tumor eradication is small and even harder to achieve in 3D. The lower efficiency in 3D exists despite the presence of many more adjacent cells in the 3D environment that results in a shorter time to reach equilibrium. The mean field mathematical models generally used to describe tumor virotherapy appear to provide an overoptimistic view of the outcomes of therapy. Three dimensional space provides a significant barrier to efficient and complete virus spread within tumors and needs to be explicitly taken into account for virus optimization to achieve the desired outcome of therapy.

In Vivo Imaging of Oncolytic Measles Virus Propagation with Single-Cell Resolution.

Recombinant measles viruses (MVs) have oncolytic activity against a variety of human cancers. However, their kinetics of spread within tumors has been unexplored. We established an intravital imaging system using the dorsal skin fold chamber, which allows for serial, non-invasive imaging of tumor cells and replication of a fusogenic and a hypofusogenic MV. Hypofusogenic virus-infected cells were detected at the earliest 3 days post-infection (dpi), with peak infection around 6 dpi. In contrast, the fusogenic virus replicated faster: infected cells were detectable 1 dpi and cells were killed quickly. Infection foci were significantly larger with the fusogenic virus. Both viruses formed syncytia. The spatial relationships between cells have a major influence on the outcome of therapy with oncolytic viruses.

In Vivo Estimation of Oncolytic Virus Populations within Tumors.

The use of replication-competent viruses as oncolytic agents is rapidly expanding, with several oncolytic viruses approved for cancer therapy. As responses to therapy are highly variable, understanding the dynamics of therapy is critical for optimal application of virotherapy in practice. Although mathematical models have been developed to understand the dynamics of tumor virotherapy, a scarcity of in vivo data has made difficult parametrization of these models. To tackle this problem, we studied the in vitro and in vivo spread of two oncolytic measles viruses that induce expression of the sodium iodide symporter (NIS) in cells. NIS expression enabled infected cells to concentrate radioactive isotopes that could be reproducibly and quantitatively imaged using SPECT/CT. We observed a strong linear relationship in vitro between infectious virus particles, viral N and NIS gene expression, and radioactive isotope uptake. In vivo radioisotope uptake was highly correlated with viral N and NIS gene expression. Similar expression patterns between viral N and NIS gene expression in vitro and in vivo implied that the oncolytic virus behaved similarly in both scenarios. Significant titers of viable virus were consistently isolated from tumors explanted from mice that had been injected with oncolytic measle viruses. We observed a weaker but positive in vivo relationship between radioisotope uptake and the viable virus titer recovered from tumors; this was likely due to anisotropies in the viral distribution in vivo These data suggest that methods that enable quantitation of in vivo anisotropies are required for continuing development of oncolytic virotherapy.Significance: These findings address a fundamental gap in our knowledge of oncolytic virotherapy by presenting technology that gives insight into the behavior of oncolytic viruses in vivo Cancer Res; 78(20); 5992-6000. ©2018 AACR.

Activation of Group II Metabotropic Glutamate Receptors Suppresses Excitability of Mouse Main Olfactory Bulb External Tufted and Mitral Cells.

Metabotropic glutamate receptors (mGluRs) are abundantly expressed in the rodent main olfactory bulb. The function of Group I mGluRs has been investigated in a number of studies, while the actions of Group II mGluRs, which include the mGluR2 and mGluR3 subtypes, have been less well explored. Here, we used electrophysiological approaches in mouse olfactory bulb slices to investigate how Group II mGluR activation and inactivation modifies the activity of external tufted (ET) and mitral cells. The Group II mGluR agonist DCG-IV was found to directly and uniformly reduce the spontaneous discharge of ET and mitral cells. The inhibitory effect of DCG-IV was absent in mitral cells with truncated apical dendrites, indicating a glomerular site of action. DCG-IV did not influence olfactory nerve-evoked monosynaptic responses in ET or mitral cells, indicating that Group II mGluRs do not presynaptically modulate glutamate release from olfactory nerve terminals. In contrast, DCG-IV suppressed polysynaptic responses in periglomerular cells evoked by olfactory nerve stimulation. DCG-IV also inhibited glutamate release from ET cells, and suppressed the spontaneous and olfactory nerve-evoked long-lasting depolarization in mitral cells. Applied alone, Group II receptor antagonists were without effect, suggesting that basal activation of these receptors is nil. These findings suggest that Group II mGluRs inhibit ET and mitral cell activity and further dampen intraglomerular excitatory circuits by suppressing glutamate release.

Activation of ß-noradrenergic receptors enhances rhythmic bursting in mouse olfactory bulb external tufted cells.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the nucleus locus coeruleus. Despite the well-documented role of norepinephrine and ß-adrenergic receptors in neonatal odor preference learning, identified cellular physiological actions of ß-receptors in the MOB have remained elusive. ß-Receptors are expressed at relatively high levels in the MOB glomeruli, the location of external tufted (ET) cells that exert an excitatory drive on mitral and other cell types. The present study investigated the effects of ß-receptor activation on the excitability of ET cells with patch-clamp electrophysiology in mature mouse MOB slices. Isoproterenol and selective ß2-, but not ß1-, receptor agonists were found to enhance two key intrinsic currents involved in ET burst initiation: persistent sodium (INaP) and hyperpolarization-activated inward (Ih) currents. Together, the positive modulation of these currents increased the frequency and strength of ET cell rhythmic bursting. Rodent sniff frequency and locus coeruleus neuronal firing increase in response to novel stimuli or environments. The increase in ET excitability by ß-receptor activation may better enable ET cell rhythmic bursting, and hence glomerular network activity, to pace faster sniff rates during heightened norepinephrine release associated with arousal.

Increased olfactory bulb acetylcholine bi-directionally modulates glomerular odor sensitivity.

The glomerular layer of the olfactory bulb (OB) receives heavy cholinergic input from the horizontal limb of the diagonal band of Broca (HDB) and expresses both muscarinic and nicotinic acetylcholine (ACh) receptors. However, the effects of ACh on OB glomerular odor responses remain unknown. Using calcium imaging in transgenic mice expressing the calcium indicator GCaMP2 in the mitral/tufted cells, we investigated the effect of ACh on the glomerular responses to increasing odor concentrations. Using HDB electrical stimulation and in vivo pharmacology, we find that increased OB ACh leads to dynamic, activity-dependent bi-directional modulation of glomerular odor response due to the combinatorial effects of both muscarinic and nicotinic activation. Using pharmacological manipulation to reveal the individual receptor type contributions, we find that m2 muscarinic receptor activation increases glomerular sensitivity to weak odor input whereas nicotinic receptor activation decreases sensitivity to strong input. Overall, we found that ACh in the OB increases glomerular sensitivity to odors and decreases activation thresholds. This effect, along with the decreased responses to strong odor input, reduces the response intensity range of individual glomeruli to increasing concentration making them more similar across the entire concentration range. As a result, odor representations are more similar as concentration increases.

Computational modeling suggests distinct, location-specific function of norepinephrine in olfactory bulb and piriform cortex.

Noradrenergic modulation from the locus coerulus is often associated with the regulation of sensory signal-to-noise ratio. In the olfactory system, noradrenergic modulation affects both bulbar and cortical processing, and has been shown to modulate the detection of low concentration stimuli. We here implemented a computational model of the olfactory bulb and piriform cortex, based on known experimental results, to explore how noradrenergic modulation in the olfactory bulb and piriform cortex interact to regulate odor processing. We show that as predicted by behavioral experiments in our lab, norepinephrine can play a critical role in modulating the detection and associative learning of very low odor concentrations. Our simulations show that bulbar norepinephrine serves to pre-process odor representations to facilitate cortical learning, but not recall. We observe the typical non-uniform dose-response functions described for norepinephrine modulation and show that these are imposed mainly by bulbar, but not cortical processing.

Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

Activation of group I metabotropic glutamate receptors enhances persistent sodium current and rhythmic bursting in main olfactory bulb external tufted cells.

Rhythmically bursting olfactory bulb external tufted (ET) cells are thought to play a key role in synchronizing glomerular network activity to respiratory-driven sensory input. Whereas spontaneous bursting in these cells is intrinsically generated by interplay of several voltage-dependent currents, bursting strength and frequency can be modified by local intrinsic and centrifugal synaptic input. Activation of metabotropic glutamate receptors (mGluRs) engages a calcium-dependent cation current (I(CAN)) that increases rhythmic bursting, but mGluRs may also modulate intrinsic mechanisms involved in bursting. Here, we used patch-clamp electrophysiology in rat olfactory bulb slices to investigate whether mGluRs modulate two key intrinsic currents involved in ET cell burst initiation: persistent sodium (I(NaP)) and hyperpolarization-activated cation (Ih) currents. Using a BAPTA-based internal solution to block I(CAN), we found that the mGluR1/5 agonist DHPG enhanced I(NaP) but did not alter Ih. I(NaP) enhancement consisted of increased current at membrane potentials between -60 and -50 mV and a hyperpolarizing shift in activation threshold. Both effects would be predicted to shorten the interburst interval. In agreement, DHPG modestly depolarized (∼3.5 mV) ET cells and increased burst frequency without effect on other major burst parameters. This increase was inversely proportional to the basal burst rate such that slower ET cells exhibited the largest increases. This may enable ET cells with slow intrinsic burst rates to pace with faster sniff rates. Taken with other findings, these results indicate that multiple neurotransmitter mechanisms are engaged to fine-tune rhythmic ET cell bursting to context- and state-dependent changes in sniffing frequency.

Noradrenergic but not cholinergic modulation of olfactory bulb during processing of near threshold concentration stimuli.

Neuromodulatory systems such as noradrenaline (NE), acetylcholine (ACh), and serotonin (5HT) serve important functions in sensory perception. We use the olfactory bulb (OB) as a model system to study the roles of individual neuromodulators in sensory perception. Using a spontaneous, nonreward motivated detection task, as well as a reward-motivated task, we show that rats can easily respond to odorants at very low concentrations when motivated to do so in a food-rewarded task, despite not showing spontaneous responses to these low concentration odorants. Using the same tasks paired with local bulbar infusions of noradrenergic and cholinergic drugs, we then show that rats engage their noradrenergic, but not their cholinergic system, to better respond to near threshold odorants. These results suggest that while cholinergic modulation of OB function is mostly important for odor decorrelation and discrimination, noradrenergic modulation is important for signal-to-noise modulation.

Expression of transient receptor potential (TRP) channel mRNAs in the mouse olfactory bulb.

Transient receptor potential (TRP) channels are a large family of cation channels. The 28 TRP channel subtypes in rodent are divided into 6 subfamilies: TRPC1-7, TRPV1-6, TRPM1-8, TRPP2/3/5, TRPML1-3 and TRPA1. TRP channels are involved in peripheral olfactory transduction. Several TRPC channels are expressed in unidentified neurons in the main olfactory bulb (OB), but the expression of most TRP channels in the OB has not been investigated. The present study employed RT-PCR as an initial survey of the expression of TRP channel mRNAs in the mouse OB and in 3 cell types: external tufted, mitral and granule cells. All TRP channel mRNAs except TRPV5 were detected in OB tissue. Single cell RT-PCR revealed that external tufted, mitral and granule cell populations expressed in aggregate 14 TRP channel mRNAs encompassing members of all 6 subfamilies. These different OB neuron populations expressed 7-12 channel mRNAs. Common channel expression was more similar among external tufted and mitral cells than among these cells and granule cells. These results indicate that a large number of TRP channel subtypes are expressed in OB neurons, providing the molecular bases for these channels to regulate OB neuron activity and central olfactory processing.

Cdc42 and the guanine nucleotide exchange factors Ect2 and trio mediate Fn14-induced migration and invasion of glioblastoma cells.

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor-inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14-directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy.

Nonlinear effects of noradrenergic modulation of olfactory bulb function in adult rodents.

The mammalian main olfactory bulb receives a significant noradrenergic input from the locus coeruleus. Norepinephrine (NE) is involved in acquisition of conditioned odor preferences in neonatal animals, in some species-specific odor-dependent behaviors, and in adult odor perception. We provide a detailed review of the functional role of NE in adult rodent main olfactory bulb function. We include cellular, synaptic, network, and behavioral data and use computational simulations to tie these different types of data together.

TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling.

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.

Mutations in the stalk region of the measles virus hemagglutinin inhibit syncytium formation but not virus entry.

Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.

Noradrenergic modulation of behavioral odor detection and discrimination thresholds in the olfactory bulb.

The mammalian main olfactory bulb (MOB) receives a significant noradrenergic input from the locus coeruleus. Norepinephrine (NE) is involved in the acquisition of conditioned odor preferences in neonatal animals and in some species-specific odor-dependent behaviors. Thus far, the role of NE in odor processing in adult rats remains less studied. We investigated the role of noradrenergic modulation in the MOB on odor detection and discrimination thresholds using behavioral and computational modeling approaches. Adult rats received bilateral MOB injections of vehicle, NE (0.1-1000 microM), noradrenergic receptor antagonists and NE + receptor antagonists combined. NE infusion improved odor detection and discrimination as a function of NE and odor concentration. The effect of NE on detection and discrimination magnitude at any given odor concentration varied in a non-linear function with respect to NE concentration. Receptor antagonist infusion demonstrated that alpha1 receptor activation is necessary for the modulatory effect of NE. Computational modeling showed that increases in the strength of alpha1 receptor activation leads to improved odor signal-to-noise ratio and spike synchronization in mitral cells that may underlie the behaviorally observed decrease of detection and discrimination thresholds. Our results are the first to show that direct infusion of NE or noradrenergic receptor antagonists into a primary sensory network modulates sensory detection and discrimination thresholds at very low stimulus concentrations.

Effects of neonatal inflammation on descending modulation from the rostroventromedial medulla.

Cutaneous tissue inflammation during the first postnatal week is known to alter long-term development of spinal cord nociceptive circuitry and to alter behavioral responses to noxious stimuli in adult animals. The impact of neonatal inflammation on descending projections arising from supraspinal sites that modulate spinal nociceptive processing is unknown. In the present study, we investigated if altered behavioral responses to pain in adult animals after neonatal inflammation are associated with changes in descending modulation of nocifensive responses elicited from the rostroventromedial medulla (RVM) in lightly anesthetized rats. Compared to handled control animals, hindpaw injection of 0.25% carrageenan (CG) at postnatal day 3 produced adult basal hypoalgesia and increased hyperalgesia 24 h after reinflammation with Complete Freund's Adjuvant (CFA) in awake animals. These effects were specific to the neonatally treated hindpaw, partially replicating previous findings, but were absent in lightly anesthetized animals. However, focal electrical stimulation of the RVM in lightly anesthetized CG treated animals produced significantly greater descending inhibition of nocifensive responses to noxious thermal stimuli applied to the hindpaws and the tail. These effects were partially replicated by intra-RVM microinjection of AMPA. No differences in the efficacy of RVM stimulation between CG and control animals were observed 24h after reinflammation with CFA. These findings indicate that neonatal tissue injury and inflammation produces lasting alterations in descending modulatory systems that modify nociceptive processing. Taken together with previous studies, these results indicate that changes in pain sensitivity following neonatal tissue injury involve long-term alterations in spinal and supraspinal circuitry.

In silico evolutionary dynamics of tumour virotherapy.

Replication-competent viruses based on the Edmonston vaccine strain of measles virus (MV-Edm) have potent and selective activities against various types of tumours in vitro but the responses in vivo are more variable. Some tumours are eliminated consistently while others persist despite evidence of ongoing viral propagation. In order to understand these disparate results, we have developed a model for the spatial growth of a tumour population followed by infection with a replicating virus that can spread by cell-to-cell fusion ultimately leading to cell death. We utilize the model to explore both the impact of tumour architecture and the dynamics of tumour cell-virus interactions on the outcome of therapy.