Transient receptor potential Ankyrin-1 (TRPA1) agonists suppress myelination and induce demyelination in organotypic cortical slices.

Oligodendrocytes are highly specialized glial cells characterized by their production of multilayer myelin sheaths that wrap axons to speed up action potential propagation. It is due to their specific role in supporting axons that impairment of myelin structure and function leads to debilitating symptoms in a wide range of degenerative diseases, including Multiple Sclerosis and Leukodystrophies. It is known that myelin damage can be receptor-mediated and recently oligodendrocytes have been shown to express Ca2+ -permeable Transient Receptor Potential Ankyrin-1 (TRPA1) channels, whose activation can result in myelin damage in ischemia. Here, we show, using organotypic cortical slice cultures, that TRPA1 activation, by TRPA1 agonists JT010 and Carvacrol for varying lengths of time, induces myelin damage. Although TRPA1 activation does not appear to affect oligodendrocyte progenitor cell number or proliferation, it prevents myelin formation and after myelination causes internodal shrinking and significant myelin degradation. This does not occur when the TRPA1 antagonist, A967079, is also applied. Of note is that when TRPA1 agonists are applied for either 24 h, 3 days or 7 days, axon integrity appears to be preserved while mature myelinated oligodendrocytes remain but with significantly shortened internodes. These results provide further evidence that TRPA1 inhibition could be protective in demyelination diseases and a promising therapy to prevent demyelination and promote remyelination.

Proton-gated Ca(2+)-permeable TRP channels damage myelin in conditions mimicking ischaemia.

The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. In ischaemia myelin is damaged in a Ca(2+)-dependent manner, abolishing action potential propagation. This has been attributed to glutamate release activating Ca(2+)-permeable N-methyl-D-aspartate (NMDA) receptors. Surprisingly, we now show that NMDA does not raise the intracellular Ca(2+) concentration ([Ca(2+)]i) in mature oligodendrocytes and that, although ischaemia evokes a glutamate-triggered membrane current, this is generated by a rise of extracellular [K(+)] and decrease of membrane K(+) conductance. Nevertheless, ischaemia raises oligodendrocyte [Ca(2+)]i, [Mg(2+)]i and [H(+)]i, and buffering intracellular pH reduces the [Ca(2+)]i and [Mg(2+)]i increases, showing that these are evoked by the rise of [H(+)]i. The H(+)-gated [Ca(2+)]i elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl pyrophosphate, HC-030031, A967079 or TRPA1 knockout. TRPA1 block reduces myelin damage in ischaemia. These data suggest that TRPA1-containing ion channels could be a therapeutic target in white matter ischaemia.

Prevention and management of allergic reactions to food in child care centers and schools: Practice guidelines.

Food allergy management in child care centers and schools is a controversial topic, for which evidence-based guidance is needed. Following the Grading of Recommendations Assessment, Development, and Evaluation approach, we conducted systematic literature reviews of the anticipated health effects of selected interventions for managing food allergy in child care centers and schools; we compiled data about the costs, feasibility, acceptability, and effects on health equity of the selected interventions; and we developed the following conditional recommendations: we suggest that child care centers and schools implement allergy training and action plans; we suggest that they use epinephrine (adrenaline) to treat suspected anaphylaxis; we suggest that they stock unassigned epinephrine autoinjectors, instead of requiring students to supply their own personal autoinjectors to be stored on site for designated at-school use; and we suggest that they do not implement site-wide food prohibitions (eg, "nut-free" schools) or allergen-restricted zones (eg, "milk-free" tables), except in the special circumstances identified in this document. The recommendations are labeled "conditional" due to the low quality of available evidence. More research is needed to determine with greater certainty which interventions are likely to be the most beneficial. Policymakers might need to adapt the recommendations to fit local circumstances.

Effects of the ecto-ATPase apyrase on microglial ramification and surveillance reflect cell depolarization, not ATP depletion.

Microglia, the brain's innate immune cells, have highly motile processes which constantly survey the brain to detect infection, remove dying cells, and prune synapses during brain development. ATP released by tissue damage is known to attract microglial processes, but it is controversial whether an ambient level of ATP is needed to promote constant microglial surveillance in the normal brain. Applying the ATPase apyrase, an enzyme which hydrolyzes ATP and ADP, reduces microglial process ramification and surveillance, suggesting that ambient ATP/ADP maintains microglial surveillance. However, attempting to raise the level of ATP/ADP by blocking the endogenous ecto-ATPase (termed NTPDase1/CD39), which also hydrolyzes ATP/ADP, does not affect the cells' ramification or surveillance, nor their membrane currents, which respond to even small rises of extracellular [ATP] or [ADP] with the activation of K+ channels. This indicates a lack of detectable ambient ATP/ADP and ecto-ATPase activity, contradicting the results with apyrase. We resolve this contradiction by demonstrating that contamination of commercially available apyrase by a high K+ concentration reduces ramification and surveillance by depolarizing microglia. Exposure to the same K+ concentration (without apyrase added) reduced ramification and surveillance as with apyrase. Dialysis of apyrase to remove K+ retained its ATP-hydrolyzing activity but abolished the microglial depolarization and decrease of ramification produced by the undialyzed enzyme. Thus, applying apyrase affects microglia by an action independent of ATP, and no ambient purinergic signaling is required to maintain microglial ramification and surveillance. These results also have implications for hundreds of prior studies that employed apyrase to hydrolyze ATP/ADP.

Capillary pericytes regulate cerebral blood flow in health and disease.

Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.

Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure.

Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

Endogenous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length.

Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, using organotypic cerebral cortex slices from mice expressing eGFP in Sox10-positive oligodendrocytes, that endogenously released GABA, acting on GABAA receptors, greatly reduces the number of oligodendrocyte lineage cells. The decrease in oligodendrocyte number correlates with a reduction in the amount of myelination but also an increase in internode length, a parameter previously thought to be set by the axon diameter or to be a property intrinsic to oligodendrocytes. Importantly, while TTX block of neuronal activity had no effect on oligodendrocyte lineage cell number when applied alone, it was able to completely abolish the effect of blocking GABAA receptors, suggesting that control of myelination by endogenous GABA may require a permissive factor to be released from axons. In contrast, block of AMPA/KA receptors had no effect on oligodendrocyte lineage cell number or myelination. These results imply that, during development, GABA can act as a local environmental cue to control myelination and thus influence the conduction velocity of action potentials within the CNS. GLIA 2017;65:309-321.

Do astrocytes really exocytose neurotransmitters?

In the past 20 years, an extra layer of information processing, in addition to that provided by neurons, has been proposed for the CNS. Neuronally evoked increases of the intracellular calcium concentration in astrocytes have been suggested to trigger exocytotic release of the 'gliotransmitters' glutamate, ATP and D-serine. These are proposed to modulate neuronal excitability and transmitter release, and to have a role in diseases as diverse as stroke, epilepsy, schizophrenia, Alzheimer's disease and HIV infection. However, there is intense controversy about whether astrocytes can exocytose transmitters in vivo. Resolving this issue would considerably advance our understanding of brain function.

Properties and fate of oligodendrocyte progenitor cells in the corpus callosum, motor cortex, and piriform cortex of the mouse.

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP(+)) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2'-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2(+) OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na(+) channels (I(Na)). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2(+) NG2(+) I(Na)(+) OPCs and OLIG2(+) NG2(neg) I(Na)(neg) OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced I(Na) and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα-CreER(T2) : R26R-YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.

A phase 1 study evaluating the combination of an allosteric AKT inhibitor (MK-2206) and trastuzumab in patients with HER2-positive solid tumors.

INTRODUCTION: Trastuzumab is effective in human epidermal growth factor receptor 2 (HER2)-over-expressing breast and gastric cancers. However, patients may develop resistance through downstream signaling via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. This phase 1 trial was conducted to determine the safety and tolerability of the investigational AKT inhibitor MK-2206, to prepare for future studies to determine whether the combination with trastuzumab could inhibit compensatory signaling. METHODS: Patients with HER2+ treatment-refractory breast and gastroesophageal cancer were enrolled. Treatment consisted of standard doses of intravenous trastuzumab and escalating dose levels of oral MK-2206 using either an every-other-day (45 mg and 60 mg QOD) or once-weekly (135 mg and 200 mg QW) schedule. RESULTS: A total of 34 patients with HER2+ disease were enrolled; 31 received study-drug. The maximum tolerated dose (MTD) for MK-2206 in combination with trastuzumab was 60 mg for the QOD schedule and 135 mg for the QW schedule, although a true MTD was not established due to early termination of the trial. The most common treatment-emergent toxicities included fatigue, hyperglycemia, and dermatologic rash, consistent with prior experience; one death unrelated to treatment was reported. There was one complete response in a patient with metastatic breast cancer, one patient achieved a partial response, and 5 patients had stable disease for at least 4 months, despite progression on multiple prior trastuzumab- and lapatinib-based therapies. Results also indicate that trastuzumab does not affect the pharmacokinetics of MK-2206. CONCLUSIONS: Results suggest the AKT inhibitor MK-2206 can be safely combined with trastuzumab, and is associated with clinical activity, supporting further investigation. TRIAL REGISTRATION: ClinicalTrials.gov; identifier: NCT00963547.

Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.

A fluorescence-based assay for the apurinic/apyrimidinic-site cleavage activity of human tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 3'-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase I-DNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 3' adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with high-throughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors.

Regulation of oligodendrocyte development and myelination by glucose and lactate.

In the gray matter of the brain, astrocytes have been suggested to export lactate (derived from glucose or glycogen) to neurons to power their mitochondria. In the white matter, lactate can support axon function in conditions of energy deprivation, but it is not known whether lactate acts by preserving energy levels in axons or in oligodendrocytes, the myelinating processes of which are damaged rapidly in low energy conditions. Studies of cultured cells suggest that oligodendrocytes are the cell type in the brain that consumes lactate at the highest rate, in part to produce membrane lipids presumably for myelin. Here, we use pH imaging to show that oligodendrocytes in the white matter of the rat cerebellum and corpus callosum take up lactate via monocarboxylate transporters (MCTs), which we identify as MCT1 by confocal immunofluorescence and electron microscopy. Using cultured slices of developing cerebral cortex from mice in which oligodendrocyte lineage cells express GFP (green fluorescent protein) under the control of the Sox10 promoter, we show that a low glucose concentration reduces the number of oligodendrocyte lineage cells and myelination. Myelination is rescued when exogenous l-lactate is supplied. Thus, lactate can support oligodendrocyte development and myelination. In CNS diseases involving energy deprivation at times of myelination or remyelination, such as periventricular leukomalacia leading to cerebral palsy, stroke, and secondary ischemia after spinal cord injury, lactate transporters in oligodendrocytes may play an important role in minimizing the inhibition of myelination that occurs.

Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.

A defining feature of glial cells has been their inability to generate action potentials. We show here that there are two distinct types of morphologically identical oligodendrocyte precursor glial cells (OPCs) in situ in rat CNS white matter. One type expresses voltage-gated sodium and potassium channels, generates action potentials when depolarized and senses its environment by receiving excitatory and inhibitory synaptic input from axons. The other type lacks action potentials and synaptic input. We found that when OPCs suffered glutamate-mediated damage, as occurs in cerebral palsy, stroke and spinal cord injury, the action potential-generating OPCs were preferentially damaged, as they expressed more glutamate receptors, and received increased spontaneous glutamatergic synaptic input in ischemia. These data challenge the idea that only neurons generate action potentials in the CNS and imply that the development of therapies for demyelinating disorders will require defining which OPC type can carry out remyelination.

Transient Receptor Potential Ankyrin-1 (TRPA1) Block Protects against Loss of White Matter Function during Ischaemia in the Mouse Optic Nerve.

Oligodendrocytes produce myelin, which provides insulation to axons and speeds up neuronal transmission. In ischaemic conditions, myelin is damaged, resulting in mental and physical disabilities. Recent evidence suggests that oligodendrocyte damage during ischaemia can be mediated by Transient Receptor Potential Ankyrin-1 (TRPA1), whose activation raises intracellular Ca2+ concentrations and damages compact myelin. Here, we show that TRPA1 is constitutively active in oligodendrocytes and the optic nerve, as the specific TRPA1 antagonist, A-967079, decreases basal oligodendrocyte Ca2+ concentrations and increases the size of the compound action potential (CAP). Conversely, TRPA1 agonists reduce the size of the optic nerve CAP in an A-967079-sensitive manner. These results indicate that glial TRPA1 regulates neuronal excitability in the white matter under physiological as well as pathological conditions. Importantly, we find that inhibition of TRPA1 prevents loss of CAPs during oxygen and glucose deprivation (OGD) and improves the recovery. TRPA1 block was effective when applied before, during, or after OGD, indicating that the TRPA1-mediated damage is occurring during both ischaemia and recovery, but importantly, that therapeutic intervention is possible after the ischaemic insult. These results indicate that TRPA1 has an important role in the brain, and that its block may be effective in treating many white matter diseases.

Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2-glia.

NG2-glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2-glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12-16, after the main period of oligodendrocyte generation. Using fluo-4 and fura-2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca(2+) signals in NG2-glia in situ, acting on AMPA-type glutamate receptors and P2Y(1) and P2X(7) purine receptors; NMDA evoked a weak Ca(2+) signal in a small proportion of NG2-glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2-glia; ATP alone evokes robust Ca(2+) signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2-glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2-glia and astrocytes. In addition, we provide evidence that NG2-glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2-glia within a neuron-glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons.

The effect of N-acetyl-aspartyl-glutamate and N-acetyl-aspartate on white matter oligodendrocytes.

Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavan's disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca(2+)](i) in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies.

Effects of glutamate receptor activation on NG2-glia in the rat optic nerve.

NG2-glia are a substantial population of cells in the central nervous system (CNS) that can be identified by their specific expression of the NG2 chondroitin sulphate (CSPG). NG2-glia can generate oligodendrocytes, but it is unlikely this is their only function; indeed, they may be multipotent neural stem cells. Moreover, NG2-glia are a highly reactive cell type and a major function is to help form the axon growth inhibitory glial scar in response to CNS injury. The factors that regulate these diverse behaviours of NG2-glia are not fully resolved, but NG2-glia express receptors to the neurotransmitter glutamate, which has known potent effects on other glia. Here, we have examined the actions of glutamate receptor activation on NG2-glia in the rat optic nerve, a typical CNS white matter tract that does not contain neuronal cell bodies. Glutamate induces an increase in [Ca(2+)](i) in immuno-identified NG2-glia in situ and in vitro. In addition, we examined the effects of glutamate receptor activation in vivo by focal injection of the glutamate receptor agonist kainate into the optic nerve; saline was injected in controls. Changes in glial and axonal function were determined at 7 days post injection (dpi), by immunohistochemistry and electrophysiological measurement of the compound action potential (CAP). Injection of kainate resulted in a highly localized 'injury response' in NG2-glia, marked by dense labelling for NG2 at the lesion site, as compared to astrocytes, which displayed a more extensive reactive astrogliosis. Furthermore, injection of kainate resulted in an axonal conduction block. These glial and axonal changes were not observed following injection of saline vehicle. In addition, we provide evidence that endogenous glutamate induces calcium-dependent phosphorylation of extracellular signal-regulated kinases (ERK1/2), which may provide a potential mechanism by which glutamate-mediated changes in raised intracellular calcium could regulate the observed gliosis. The results provide evidence that activation of AMPA-kainate type ionotropic glutamate receptors evoke raised calcium in NG2-glia and induces an injury response in NG2-glia.

The role of TRP channels in white matter function and ischaemia.

Transient receptor potential (TRP) proteins are a large family of tetrameric non-selective cation channels that are widely expressed in the grey and white matter of the CNS, and are increasingly considered as potential therapeutic targets in brain disorders. Here we briefly review the evidence for TRP channel expression in glial cells and their possible role in both glial cell physiology and stroke. Despite their contribution to important functions, our understanding of the roles of TRP channels in glia is still in its infancy. The evidence reviewed here indicates that further investigation is needed to determine whether TRP channel inhibition can decrease damage or increase repair in stroke and other diseases affecting the white matter.