Patricia J. Armati

Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN), play a role in the pathogenesis of neurological diseases, in particular AIDS dementia complex. Although QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the role of astrocytes in QUIN production is controversial. Using cytokine‐stimulated cultures of human astrocytes, we assayed key enzymes and products of the KP. We found that human astrocytes lack kynurenine hydroxylase so that large amounts of kynurenine and the QUIN antagonist kynurenic acid were produced. However, the amounts of QUIN that were synthesized were subsequently completely degraded. We then showed that kynurenine in concentrations comparable with those produced by astrocytes led to significant production of QUIN by macrophages. These results suggest that astrocytes alone are neuroprotective by minimizing QUIN production and maximizing synthesis of kynurenic acid. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of large concentrations of kynurenine that can be secondarily metabolized by neighbouring or infiltrating monocytic cells to form the neurotoxin QUIN.

Expression of the kynurenine pathway enzymes in human microglia and macrophages.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN) play a role in the pathogenesis of neurological diseases. Monocytic cells are known to be the major producers of QUIN. However, macrophages have the ability to produce approximately 20 to 30-fold more QUIN than microglia. The molecular origin of this difference has not been clarified yet. Using unstimulated and IFN-gamma-stimulated cultures of human fcetal microglia and adult macrophages, we assayed mRNA expression of 8 key enzymes of the KP using RT-PCR and QUIN production using GC-MS. We found that after stimulation with IFN-gamma microglia produced de novo 20-fold less QUIN than macrophages. This quantitative difference in the ability to produce QUIN appears to be associated with a lower expression of 3 important enzymes of the KP in microglia: indoleamine 2,3-dioxygenase (IDO), kynureninase (KYNase) and kynurenine hydroxylase (KYN(OH)ase). These results suggest that activated infiltrating macrophages are the most potent QUIN producers during brain inflammatory diseases with playing a lesser role.

Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCε-mediated phosphorylation at S800

Abstract Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1‐mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+‐imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+‐imaging and patch‐clamp methods, we show that PMA‐induced activation of PKC&egr; is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKC&egr;‐mediated phosphorylation. Patch‐clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non‐specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKC&egr; paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti‐phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKC&egr; and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

Herpes Simplex Virus Tegument Protein US11 Interacts with Conventional Kinesin Heavy Chain

ABSTRACT Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons.

Anterograde Transport of Herpes Simplex Virus Type 1 in Cultured, Dissociated Human and Rat Dorsal Root Ganglion Neurons

ABSTRACT The mechanism of anterograde transport of herpes simplex virus was studied in cultured dissociated human and rat dorsal root ganglion neurons. The neurons were infected with HSV-1 to examine the distribution of capsid (VP5), tegument (VP16), and glycoproteins (gC and gB) at 2, 6, 10, 13, 17, and 24 h postinfection (p.i.) with or without nocodazole (a microtubule depolymerizer) or brefeldin A (a Golgi inhibitor). Retrogradely transported VP5 was detected in the cytoplasm of the cell body up to the nuclear membrane at 2 h p.i. It was first detected de novo in the nucleus and cytoplasm at 10 h p.i., the axon hillock at 13 h p.i., and the axon at 15 to 17 h p.i. gC and gB were first detected de novo in the cytoplasm and the axon hillock at 10 h p.i. and then in the axon at 13 h p.i., which was always earlier than the detection of VP5. De novo-synthesized VP16 was first detected in the cytoplasm at 10 to 13 h p.i. and in the axon at 16 to 17 h p.i. Nocodazole inhibited the transport of all antigens, VP5, VP16, and gC or gB. The kinetics of inhibition of VP5 and gC could be dissociated. Brefeldin A inhibited the transport of gC or gB and VP16 but not VP5 into axons. Transmission immunoelectron microscopy confirmed that there were unenveloped nucleocapsids in the axon with or without brefeldin A. These findings demonstrate that glycoproteins and capsids, associated with tegument proteins, are transported by different pathways with slightly differing kinetics from the nucleus to the axon. Furthermore, axonal anterograde transport of the nucleocapsid can proceed despite the loss of most VP16.

830 nm laser irradiation induces varicosity formation, reduces mitochondrial membrane potential and blocks fast axonal flow in small and medium diameter rat dorsal root ganglion neurons: implications for the analgesic effects of 830 nm laser.

Abstract  We report the formation of 830 nm (cw) laser‐induced, reversible axonal varicosities, using immunostaining with β‐tubulin, in small and medium diameter, TRPV‐1 positive, cultured rat DRG neurons. Laser also induced a progressive and statistically significant decrease (p < 0.005) in MMP in mitochondria in and between static axonal varicosities. In cell bodies of the neuron, the decrease in MMP was also statistically significant (p < 0.05), but the decrease occurred more slowly. Importantly we also report for the first time that 830 nm (cw) laser blocked fast axonal flow, imaged in real time using confocal laser microscopy and JC‐1 as mitotracker.

Chronic exposure of human neurons to quinolinic acid results in neuronal changes consistent with AIDS dementia complex

Objective:Concentrations of quinolinic acid, an N-methyl-D-aspartate agonist, are often elevated for long periods of time in the cerebrospinal fluid (CSF) and brain tissue of patients with AIDS dementia complex (ADC). This study was designed to test the hypothesis that chronic exposure of human neurons to quinolinic acid levels equivalent to those in the CSF of ADC patients is neurotoxic. Design and methods:Human fetal brain 14–16 weeks post-menses was cultured in medium with no detectable levels of quinolinic acid. After 4 weeks, 350 or 1200 nmol/l quinolinic acid was added to the feeding medium for a further 5 weeks. Neurotoxicity was evaluated using immunohistochemistry, transmission and scanning electron microscopy, and image analysis. Results:A total of 1200 nmol/l quinolinic acid caused altered cell associations, a decrease in cell density and decreased microtubule-associated protein (MAP)-2 immunoreactivity compared with cultures exposed to 350 nmol/l quinolinic acid or controls. Image analysis of neurons in randomly selected fields revealed significantly swollen cells (P < 0.0001) compared with those treated with 350 nmol/l quinolinic acid or controls. Dendritic varicosities and discontinuous microtubular arrays were present in neurons exposed to both quinolinic acid concentrations, but not in control cultures. Conclusions:This study is the first to assess quinolinic acid levels in the experimental medium, and demonstrates that chronic exposure of human neurons to concentrations of quinolinic acid equivalent to those in the CSF of patients with ADC leads to alterations in dendritic ultrastructure and MAP-2 immunoreactivity, which is consistent with ADC pathology.

Inhibitory Effects of Laser Irradiation on Peripheral Mammalian Nerves and Relevance to Analgesic Effects: A Systematic Review

OBJECTIVE The objective of this review was to systematically identify experimental studies of non-ablative laser irradiation (LI) on peripheral nerve morphology, physiology, and function. The findings were then evaluated with special reference to the neurophysiology of pain and implications for the analgesic effects of low-level laser therapy (LLLT). BACKGROUND LLLT is used in the treatment of pain, and laser-induced neural inhibition has been proposed as a mechanism. To date, no study has systematically evaluated the effects of LI on peripheral nerve, other than those related to nerve repair, despite the fact that experimental studies of LI on nerves have been conducted over the past 25 years. METHODS We searched computerized databases and reference lists for studies of LI effects on animal and human nerves using a priori inclusion and exclusion criteria. RESULTS We identified 44 studies suitable for inclusion. In 13 of 18 human studies, pulsed or continuous wave visible and continuous wave infrared (IR) LI slowed conduction velocity (CV) and/or reduced the amplitude of compound action potentials (CAPs). In 26 animal experiments, IR LI suppressed electrically and noxiously evoked action potentials including pro-inflammatory mediators. Disruption of microtubule arrays and fast axonal flow may underpin neural inhibition. CONCLUSIONS This review has identified a range of laser-induced inhibitory effects in diverse peripheral nerve models, which may reduce acute pain by direct inhibition of peripheral nociceptors. In chronic pain, spinal cord changes induced by LI may result in long-term depression of pain. Incomplete reporting of parameters limited aggregation of data.

Quinolinic acid upregulates chemokine production and chemokine receptor expression in astrocytes

Within the brain, quinolinic acid (QUIN) is an important neurotoxin, especially in AIDS dementia complex (ADC). Its production by monocytic lineage cells is increased in the context of inflammation. However, it is not known whether QUIN promotes inflammation. Astrocytes are important in immunoregulation within the brain and so we chose to examine the effects of QUIN on the astrocyte. Using purified primary human fetal astrocyte cultures, we determined chemokine production using ELISA assays and RT‐PCR and chemokine receptor expression using immunocytochemistry and RT‐PCR with QUIN in comparison to TNFα, IL‐1β, and IFNγ. We found that QUIN induces astrocytes to produce large quantities of MCP‐1 (CCL2) and lesser amounts of RANTES (CCL5) and IL‐8 (CXCL8). QUIN also increases SDF‐1α (CXCL12), HuMIG (CXCL9), and fractalkine (CX3CL1) mRNA expression. Moreover, QUIN leads to upregulation of the chemokine receptor expression of CXCR4, CCR5, and CCR3 in human fetal astrocytes. Most of these effects were comparable to those induced by TNFα, IL‐1β, and IFNγ. The present work represents the first evidence that QUIN induces chemokine and chemokine receptor expression in astrocytes and is at least as potent as classical mediators such as inflammatory cytokines. These results suggest that QUIN may be critical in the amplification of brain inflammation, particularly in ADC. GLIA 41:371–381, 2003.

Chronic inflammatory demyelinating polyradiculoneuropathy: from pathology to phenotype

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an inflammatory neuropathy, classically characterised by a slowly progressive onset and symmetrical, sensorimotor involvement. However, there are many phenotypic variants, suggesting that CIDP may not be a discrete disease entity but rather a spectrum of related conditions. While the abiding theory of CIDP pathogenesis is that cell-mediated and humoral mechanisms act together in an aberrant immune response to cause damage to peripheral nerves, the relative contributions of T cell and autoantibody responses remain largely undefined. In animal models of spontaneous inflammatory neuropathy, T cell responses to defined myelin antigens are responsible. In other human inflammatory neuropathies, there is evidence of antibody responses to Schwann cell, compact myelin or nodal antigens. In this review, the roles of the cellular and humoral immune systems in the pathogenesis of CIDP will be discussed. In time, it is anticipated that delineation of clinical phenotypes and the underlying disease mechanisms might help guide diagnostic and individualised treatment strategies for CIDP.