Effect of Cannabidiol on Drop Seizures in the Lennox-Gastaut Syndrome.

BACKGROUND: Cannabidiol has been used for treatment-resistant seizures in patients with severe early-onset epilepsy. We investigated the efficacy and safety of cannabidiol added to a regimen of conventional antiepileptic medication to treat drop seizures in patients with the Lennox-Gastaut syndrome, a severe developmental epileptic encephalopathy. METHODS: In this double-blind, placebo-controlled trial conducted at 30 clinical centers, we randomly assigned patients with the Lennox-Gastaut syndrome (age range, 2 to 55 years) who had had two or more drop seizures per week during a 28-day baseline period to receive cannabidiol oral solution at a dose of either 20 mg per kilogram of body weight (20-mg cannabidiol group) or 10 mg per kilogram (10-mg cannabidiol group) or matching placebo, administered in two equally divided doses daily for 14 weeks. The primary outcome was the percentage change from baseline in the frequency of drop seizures (average per 28 days) during the treatment period. RESULTS: A total of 225 patients were enrolled; 76 patients were assigned to the 20-mg cannabidiol group, 73 to the 10-mg cannabidiol group, and 76 to the placebo group. During the 28-day baseline period, the median number of drop seizures was 85 in all trial groups combined. The median percent reduction from baseline in drop-seizure frequency during the treatment period was 41.9% in the 20-mg cannabidiol group, 37.2% in the 10-mg cannabidiol group, and 17.2% in the placebo group (P=0.005 for the 20-mg cannabidiol group vs. placebo group, and P=0.002 for the 10-mg cannabidiol group vs. placebo group). The most common adverse events among the patients in the cannabidiol groups were somnolence, decreased appetite, and diarrhea; these events occurred more frequently in the higher-dose group. Six patients in the 20-mg cannabidiol group and 1 patient in the 10-mg cannabidiol group discontinued the trial medication because of adverse events and were withdrawn from the trial. Fourteen patients who received cannabidiol (9%) had elevated liver aminotransferase concentrations. CONCLUSIONS: Among children and adults with the Lennox-Gastaut syndrome, the addition of cannabidiol at a dose of 10 mg or 20 mg per kilogram per day to a conventional antiepileptic regimen resulted in greater reductions in the frequency of drop seizures than placebo. Adverse events with cannabidiol included elevated liver aminotransferase concentrations. (Funded by GW Pharmaceuticals; GWPCARE3 ClinicalTrials.gov number, NCT02224560 .).

Randomized, dose-ranging safety trial of cannabidiol in Dravet syndrome.

OBJECTIVE: To evaluate the safety and preliminary pharmacokinetics of a pharmaceutical formulation of purified cannabidiol (CBD) in children with Dravet syndrome. METHODS: Patients aged 4-10 years were randomized 4:1 to CBD (5, 10, or 20 mg/kg/d) or placebo taken twice daily. The double-blind trial comprised 4-week baseline, 3-week treatment (including titration), 10-day taper, and 4-week follow-up periods. Completers could continue in an open-label extension. Multiple pharmacokinetic blood samples were taken on the first day of dosing and at end of treatment for measurement of CBD, its metabolites 6-OH-CBD, 7-OH-CBD, and 7-COOH-CBD, and antiepileptic drugs (AEDs; clobazam and metabolite N-desmethylclobazam [N-CLB], valproate, levetiracetam, topiramate, and stiripentol). Safety assessments were clinical laboratory tests, physical examinations, vital signs, ECGs, adverse events (AEs), seizure frequency, and suicidality. RESULTS: Thirty-four patients were randomized (10, 8, and 9 to the 5, 10, and 20 mg/kg/d CBD groups, and 7 to placebo); 32 (94%) completed treatment. Exposure to CBD and its metabolites was dose-proportional (AUC0-t). CBD did not affect concomitant AED levels, apart from an increase in N-CLB (except in patients taking stiripentol). The most common AEs on CBD were pyrexia, somnolence, decreased appetite, sedation, vomiting, ataxia, and abnormal behavior. Six patients taking CBD and valproate developed elevated transaminases; none met criteria for drug-induced liver injury and all recovered. No other clinically relevant safety signals were observed. CONCLUSIONS: Exposure to CBD and its metabolites increased proportionally with dose. An interaction with N-CLB was observed, likely related to CBD inhibition of cytochrome P450 subtype 2C19. CBD resulted in more AEs than placebo but was generally well-tolerated. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for children with Dravet syndrome, CBD resulted in more AEs than placebo but was generally well-tolerated.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.

Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature.

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

Astrocytic activation and an inhibition of MAP kinases are required for proteinase-activated receptor-2-mediated protection from neurotoxicity.

Proteinase-activated receptor-2 (PAR-2) expression levels are altered in several CNS disorders with these changes being proposed to either exacerbate or diminish the disease state depending on the cell type in which this occurs. Here we present data investigating the consequence of PAR-2 activation on kainate (KA)-induced neurotoxicity in organotypic hippocampal slices cultures (OHSC). Exposure of OHSC to the PAR-2 activators trypsin or Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL) induced no neurotoxicity when applied alone but was neuroprotective against KA-induced neurotoxicity. SLIGRL-mediated neuroprotection involved astrocytic activation as the neuroprotective effect was abolished following OHSC pre-treatment with fluoroacetate. Moreover, co-application of either reparixin or LY341495, antagonists of the CXCR2 chemokine receptor and metabotropic glutamate receptors respectively, inhibited the SLIGRL-mediated neuroprotection. SLIGRL application inhibited both p38 MAPK and ERK activity in OHSC, but not the JNK 1/2 signalling pathway. Accordingly, the co-application of the p38 MAPK and ERK inhibitors SB203580 and UO126 reduced KA-induced cell death, mimicking PAR-2-mediated neuroprotection. These data indicate that PAR-2 activation is neuroprotective and involves astrocytic activation, gliotransmitter release, and the subsequent inhibition of MAPK signalling cascades, providing further evidence for PAR-2 as an interesting therapeutic target in certain CNS disorders.

Rapid dendritic and axonal responses to neuronal insults.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system playing critical roles in basal synaptic transmission and mechanisms of learning and memory. Under normal conditions, glutamate is sequestered within synaptic vesicles (approximately 100 mM) with extracellular glutamate concentrations being limited (<1 microM), via retrieval by plasma-membrane transporters on neuronal and glial cells. In the case of central nervous system trauma, stroke, epilepsy, and in certain neurodegenerative diseases, increased concentrations of extracellular glutamate (by vesicular release, cell lysis and/or decreased glutamate transporter uptake/reversal) stimulate the overactivation of local ionotropic glutamate receptors that trigger neuronal cell death (excitotoxicity). Other natural agonists, such as domoic acid, alcohol and auto-antibodies, have also been reported to induce excitotoxicity.

Dendritic and mitochondrial changes during glutamate excitotoxicity.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS) and is normally stored intracellularly. However, in instances of CNS injury or disease, increased concentrations of extracellular glutamate can result in the over-activation of ionotropic glutamate receptors and trigger neuronal cell death (termed excitotoxicity). Two early hallmarks of such neuronal toxicity are mitochondrial dysfunction (depolarisation, decreased ATP synthesis, structural collapse and potential opening of the permeability transition pore) and the formation of focal swellings (also termed varicosities/beads) along the length of the dendrites. In this review, we summarise current knowledge of the mechanisms that underlie these early excitotoxic events as well as the mechanisms that facilitate dendritic recovery following termination of the excitotoxic insult.

Mitochondrial dysfunction and dendritic beading during neuronal toxicity.

Mitochondrial dysfunction (depolarization and structural collapse), cytosolic ATP depletion, and neuritic beading are early hallmarks of neuronal toxicity induced in a variety of pathological conditions. We show that, following global exposure to glutamate, mitochondrial changes are spatially and temporally coincident with dendritic bead formation. During oxygen-glucose deprivation, mitochondrial depolarization precedes mitochondrial collapse, which in turn is followed by dendritic beading. These events travel as a wave of activity from distal dendrites toward the neuronal cell body. Despite the spatiotemporal relationship between dysfunctional mitochondria and dendritic beads, mitochondrial depolarization and cytoplasmic ATP depletion do not trigger these events. However, mitochondrial dysfunction increases neuronal vulnerability to these morphological changes during normal physiological activity. Our findings support a mechanism whereby, during glutamate excitotoxicity, Ca(2+) influx leads to mitochondrial depolarization, whereas Na(+) influx leads to an unsustainable increase in ATP demand (Na(+),K(+)-ATPase activity). This leads to a drop in ATP levels, an accumulation of intracellular Na(+) ions, and the subsequent influx of water, leading to microtubule depolymerization, mitochondrial collapse, and dendritic beading. Following the removal of a glutamate challenge, dendritic recovery is dependent upon the integrity of the mitochondrial membrane potential, but not on a resumption of ATP synthesis or Na(+),K(+)-ATPase activity. Thus, dendritic recovery is not a passive reversal of the events that induce dendritic beading. These findings suggest that the degree of calcium influx and mitochondrial depolarization inflicted by a neurotoxic challenge, determines the ability of the neuron to recover its normal morphology.

Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition.

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.

PIN-G--a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins.

BACKGROUND: The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. RESULTS: Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. CONCLUSION: Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.

Cellular and subcellular calcium accumulation during glutamate-induced injury in cerebellar granule neurons.

Abstract We have investigated the role of Ca2+ accumulation and neuronal injury in cerebellar granule neurons after glutamate receptor overactivation. After the removal of the free cytosolic Ca2+ we identified an extensive second Ca2+ fraction (SCF) that is retained within the neurons after glutamate receptor overactivation. The SCF reaches a plateau within 10 min with the magnitude of this SCF accumulation reflecting the extent of the neuronal injury that occurs within the neurons. The existence of this SCF is sensitive to both NMDA receptor antagonists and mitochondrial inhibitors but is unaffected by agents that deplete endoplasmic reticulum Ca2+, indicating that this Ca2+ fraction may be located within the mitochondria. Through the isolation of mitochondria from cerebellar granule neurons treated with glutamate we have shown that the majority of the SCF is mitochondrial in location. On the removal of the glutamate stimulus the SCF recovers at a slower rate than the free Ca2+ concentration within the neuron. This is intriguing, as it implies a capacity to remember previous excitatory events. Most significantly we have shown that a short pre-application of subthreshold glutamate or kainate blocks both SCF Ca2+ accumulation and extensive neuronal injury in response to high concentrations of glutamate. These findings may be relevant to the observations of pre-conditioning in the brain and heart.

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells.

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.