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The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

We report here on a case of Ewing sarcoma (ES) occurring in a child with neurofibromatosis type 1. The sarcoma had an EWSR1-ERG translocation as well as loss of the remaining wild-type allele of NF1. Loss of the NF1 wild-type allele in the tumor suggests that activation of the Ras pathway contributed to its evolution. Review of available public data suggests that secondary mutations in the Ras pathway are found in ∼3% of ESs. This case suggests that Ras pathway activation may play a role in tumor progression in a subset of ESs.

Lower serum uric acid concentrations have been linked to cognitive dysfunction. Uric acid is an antioxidant that accounts for over half of the free radical scavenging activity in humans. Higher ser...

SummaryCopper (Cu) is an essential cofactor for a variety of metabolic functions, and the regulation of systemic Cu metabolism is critical to human health. Dietary Cu is absorbed through the intestine, stored in the liver, and mobilized into the circulation; however, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac-specific knockout of the Ctr1 Cu transporter (Ctr1hrt/hrt), resulting in cardiac Cu deficiency and severe cardiomyopathy. Unexpectedly, Ctr1hrt/hrt mice exhibited increased serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1hrt/hrt mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand, and illuminate a systemic regulation in which the Cu status of the heart is signaled to organs that take up and store Cu.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

The diagnosis, classification, and management of cancer are traditionally dictated by the site of tumor origin, for example, breast or lung, and by specific histologic subtypes of site-of-origin cancers (e.g., non–small cell versus small cell lung cancer). However, with the advent of sequencing technologies allowing for rapid, low cost, and accurate sequencing of clinical samples, new observations suggest an expanded or different approach to the diagnosis and treatment of cancer—one driven by the unique molecular features of the tumor. We discuss a genomically driven strategy for cancer treatment using BRAF as an example. Several key points are highlighted: (i) molecular aberrations can be shared across cancers; (ii) approximately 15% of all cancers harbor BRAF mutations; and (iii) BRAF inhibitors, while approved only for melanoma, have reported activity across numerous cancers and related disease types bearing BRAF aberrations. However, BRAF-mutated colorectal cancer has shown poor response rate to BRAF inhibitor monotherapy, striking a cautionary note. Yet, even in this case, emerging data suggest BRAF-mutated colorectal cancers can respond well to BRAF inhibitors, albeit when administered in combination with other agents that impact resistance pathways. Taken together, these data suggest that molecular aberrations may be the basis for a new nosology for cancer. Mol Cancer Ther; 15(4); 533–47. ©2016 AACR.

In order to identify genes involved in the development of the central nervous system (CNS) we have undertaken a gain of function screen in the embryonic CNS of Drosophila. Transposable P-elements and the UAS/GAL4 system were used to initiate transcription of genes in a pan-neural pattern using scaGAL4. Over 4100 individual P-element insertion lines were screened with monoclonal antibodies BP102 and 1D4 to visualize axon pathways. Twenty-five P-element insertions corresponding to 18 genes resulted in aberrant CNS axon pathfinding when misexpressed with scaGAL4. Genes involved in axon guidance, embryonic patterning, and cell cycle regulation were isolated. In addition, we identified several zinc finger transcription factors not previously implicated in axon guidance or CNS development. This group includes Squeeze, Kruppel homolog-1, Hepatocyte nuclear factor 4, and two uncharacterized genes, CG11966 and CG9650. Calnexin99A, a putative molecular chaperone, was isolated as well.

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase ( Abl ) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. We have identified an unusual missense ama allele, ama M109 , which dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the Ama M109 mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, we have found that all five mutations originally attributed to disabled are in fact alleles of nrt . These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone.

With the advent of genomics-based treatment in recent years, the use of targeted therapies in the treatment of various malignancies has increased exponentially. Though much data is available regarding the efficacy of targeted therapies for common malignancies, genetic cancer syndromes remain a somewhat unexplored topic with comparatively less published literature. This review seeks to characterize targeted therapy options for the following genetic cancer syndromes: Fanconi anemia, inherited medullary thyroid cancer, tuberous sclerosis, and RASopathies. By understanding the pathophysiology of these conditions as well as available molecularly targeted therapies, oncologists, in collaboration with geneticists and genetic counsellors, can begin to develop effective clinical management options and therapy regimens for the patients with these genetic syndromes that they may encounter in their practice.

Von Hippel-Lindau disease, Cowden syndrome, and Proteus syndrome are cancer syndromes which affect multiple organs and lead to significant decline in quality of life in affected patients. These syndromes are rare and typically affect the adolescent and young adult population, resulting in greater cumulative years of life lost. Improved understanding of the underpinnings of the genetic pathways underlying these syndromes and the rapid evolution of targeted therapies in general have made it possible to develop therapeutic options for these patients and other genetic cancer syndromes. Targeted therapies especially antiangiogenics and inhibitors of the PIK3CA/AKT/mTOR signaling pathway have shown activity in selected group of patients affected by these syndromes or in patients harboring specific sporadic mutations which are otherwise characteristic of these syndromes. Unfortunately due to the rare nature, patients with these syndromes are not the focus of clinical trials and unique results seen in these patients can easily go unnoticed. Most of the data suggesting benefits of targeted therapies are either case reports or small case series. Thus, a literature review was indicated. In this review we explore the use of molecularly targeted therapy options in Von Hippel-Lindau disease, Cowden syndrome, and Proteus syndrome.

Cancer genetics has rapidly evolved in the last two decades. Understanding and exploring the several genetic pathways in the cancer cell is the foundation of targeted therapy. Several genomic aberrations have been identified and their role in carcinogenesis is being explored. In contrast to most cancers where these mutations are acquired, patients with hereditary cancer syndromes have inherited genomic aberrations. The understanding of the molecular pathobiology in hereditary cancer syndromes has advanced dramatically. In addition, many molecularly targeted therapies have been developed that could have potential roles in the treatment of patients with hereditary cancer syndromes. In this review, we outline the presentation, molecular biology, and possible targeted therapies for two of the most widely recognized hereditary cancer syndromes -- hereditary breast and ovarian cancer syndrome and hereditary non-polyposis colorectal cancer syndrome (Lynch syndrome). We will also discuss other syndromes such as familial adenomatous polyposis and Li-Fraumeni syndrome (TP53).

Hereditary cancer syndromes are well known in the oncology community, typically affecting children, adolescents, and young adults and thereby resulting in great cumulative morbidity and mortality. These syndromes often lag behind their de novo counterparts in the development of approved novel treatment options due to their rarity in the general population. Recent work has allowed the identification of molecular aberrations and associated targeted therapies that may effectively treat these conditions. In this review, we seek to characterize some of the involved aberrations and associated targeted therapies for several germline malignancies, including neurofibromatosis types 1 and 2, and Gorlin syndrome. Though patients with hereditary cancer syndromes may be too rare to effectively include in large clinical trials, by understanding the pathophysiology of these diseases, clinicians can attain insights into the use of targeted therapies in their own practice when treating affected individuals.

Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.

Over the past 2 decades, the molecular mechanisms by which cells acquire, distribute, and utilize copper have been under intense investigation. Significant progress has been made in the identification of genes encoding copper homeostasis proteins and in fundamental aspects of their structure, function, and mechanisms of action in copper balance. A number of more comprehensive reviews of the field with respect to the genetics, structure, function, and physiology of copper metabolism have recently appeared elsewhere (1–4). Here, we review general mechanisms for eukaryotic copper metabolism at the cellular level in the context of recent discoveries in the field, identifying potential new functions for copper and copper metabolism proteins in cell signaling, gene expression, tumor cell metastasis, and resistance to anti-neoplastic drugs.

The BRAF serine/threonine kinase is mutated, typically at V600, to induce an active oncogenic state in a large fraction of melanoma, thyroid cancers, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers. BRAFV600E in turn activates the kinases MEK1 and MEK2, which stimulates the MAPK pathway to promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma, an effect enhanced by combining the two inhibitors. Thus, combining multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which was rescued by expressing activated MEK5 engineered to phosphorylate ERK1/2 or activated ERK2. Importantly, Cu chelators used in the treatment of Wilson disease reduced tumor growth of not only BRAFV600E-transformed cells but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers. Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, G Aaron Hobbs, Apirat Chaikuad, Stefan Knapp, Sharon L. Campbell, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4427. doi:10.1158/1538-7445.AM2014-4427

e22065 Background: A large number of esophageal cancer patients present with advanced or metastatic disease and their overall prognosis is poor. In order to improve survival, we need to identify mo...

The excitatory neurotransmitter glutamate activates various classes of ionotropic receptors in the mammalian central nervous system. αAmino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate (KA) receptor stimulation triggers Na+ influx into neurons, while N-methyl-D-aspartate (NMDA) receptor stimulation additionally leads to considerable Ca2+ influx (Hollmann and Heinemann 1994). Relative impermeability of the AMPA receptor channel to Ca2+ is regulated by the Q/R locus within the channel pore on the M2 region of the G1uR-B subunit. Expression of either glutamine (Q) or arginine (R) within this locus is controlled by posttranscriptional RNA editing (Sommer et al. 1991). Transgenic mice engineered to synthesize unedited GluR-B subunits express Ca2+permeable AMPA receptors. These mice develop progressive neurodegeneration and spontaneous seizures (Brusa et al. 1995).

The BRAF serine/threonine kinase is mutated, typically at V600, to remain in the active oncogenic state in a large fraction of melanoma, thyroid cancers, and hairy cell leukemia, and to a lesser extent in a wide spectrum of other cancers, thereby activating the kinases MEK1 and MEK2 to stimulate the MAPK pathway and promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma and early clinical studies suggest that coupling BRAF and MEK kinase inhibitors may be even more effective. Thus, the combination of multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which could be rescued by expressing activated ERK2. Importantly, a Cu chelator used in the treatment of Wilson's disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers. Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2013-4294

Parkinson's disease (PD) is characterised by the progressive degeneration of nigrostriatal dopamine (DA) neurons resulting in the major symptoms of akinesia and rigidity. Although the primary cause of PD is still not known some features make this disorder a model for neurodegenerative diseases in general. It has been known for some time that symptomatic PD can be attributed to insults with symptoms occurring many years later such as post-encephalitic PD or PD following manganese poisoning. More recently, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) has been identified as a neurotoxin selective for melanin-containing dopaminergic neurons in humans and non-human primates. The specificity of this neurotoxin and the striking clinical similarities to idiopathic PD, seen in primates, make MPTP-induced parkinsonism the most useful animal model of a neurological disease. There are numerous theoretical possibilities to interfere with both MPTP-induced neurotoxicity and the symptomatology of PD. In recent years excitatory amino acids have gained considerable interest since they can cause excitotoxic lesion of neurons under a number of pathological conditions (Olney et al., 1989; Choi, 1988). Here we summarise the present data and provide new experimental evidence indicating that MPTP-induced degeneration of dopaminergic neurons does involve glutamate-mediated toxicity. It is concluded that glutamate-mediated excitotoxicity results in the destruction of DAergic somata in the substantia nigra. Non-competitive or competitive NMDA antagonists protect nigral neurons from MPTP-induced degeneration whereas their striatal terminals still seem to degenerate.

Abecarnil is a beta-carboline agonist at benzodiazepine receptors with potent anxiolytic activity but no muscle relaxant side effects in rodents. Clinical experience suggests that changes in the muscle tone induced by benzodiazepines are related to their effects on spinal reflexes. The authors therefore analyzed the effect of treatment with abecarnil on spinal monosynaptic (Hoffmann reflexes) and polysynaptic (flexor) reflexes in mice and the influence of abecarnil on muscle tone in genetically spastic rats. The i.v. administration of abecarnil in mice (dose range, 0.02-1 mg/kg) depressed flexor reflexes in a dose-dependent manner; Hoffmann reflexes remained unchanged. Administration of diazepam i.v. (0.01-1 mg/kg) also reduced flexor reflexes and had little or no effect on Hoffmann reflexes. In genetically spastic rats, i.v. administration of abecarnil (10-30 mg/kg) decreased the muscle tone in a dose- and time-dependent manner. A similar muscle relaxant effect was observed in such rats after i.v. administration of diazepam (0.1-0.8 mg/kg). By contrast, i.p. administration of abecarnil in mice did not influence spinal reflexes up to the dose of 1 mg/kg and, in genetically spastic rats, did not affect muscle tone up to the dose of 100 mg/kg. Administration of diazepam i.p. (1 mg/kg) depressed flexor reflexes in mice and over the range 0.2 to 5 mg/kg produced a dose- and time-dependent decrease of muscle tone in genetically spastic rats. The muscle relaxant effect of i.p. diazepam could be antagonized by i.p. administered abecarnil. These studies thus demonstrate that i.v. but not i.p. administration of abecarnil may result in muscle relaxant action in mice and in genetically spastic rats.

A novel method for the assessment of the threshold for clonic seizures induced by excitatory amino acids based on continuous infusion of the glutamate agonists [alpha-amino-3-hydroxy-5-terbutyl-4-isoxazolepropionate (ATPA), kainate or N-methyl-D-aspartate (NMDA)] into the lateral brain ventricle of unrestrained mice is reported. Using this novel method of seizure threshold determination, it was found that systemically administered diphenylhydantoin and carbamazepine elevated the threshold for ATPA and had negligible effects on the threshold for kainate and NMDA. Phenobarbital and trimethadione elevated the threshold for all excitatory amino acids tested, whereas valproate elevated the threshold for ATPA and kainate seizures. Ethosuximide elevated the threshold for ATPA and kainate and decreased the threshold for NMDA seizures. The quisqualate antagonists [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine] elevated the threshold for ATPA and less so for kainate seizures, whereas the NMDA antagonist 3-((+-)2-carboxypiperazine-4-yl)-propyl-1-phosphonate elevated the threshold for NMDA seizures. 1-(4-Aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine in higher doses was also active against NMDA seizures, whereas 3-((+-)2-carboxypiperazine-4-yl)-propyl-1-phosphonate did so with kainate seizures. Among seven different convulsants, pentylenetetrazol, picrotoxin and the beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate lowered the threshold for seizures induced by excitatory amino acids. Pentylenetetrazol and picrotoxin did so with kainate seizures, whereas methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate lowered ATPA thresholds. Bicuculline, 3-mercaptopropionate, strychnine and pilocarpine were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate) is a metabolically stable anxiolytic and anticonvulsant beta-carboline derivative with few sedative and muscle relaxant effects in rodents. Abecamil binds with high affinity to benzodiazepine receptors. Because long-term treatment with benzodiazepines leads to development of dependence, we evaluated in mice whether abecarnil also possesses a potential for producing dependence, using electroencephalographic and electromyographic monitoring, and behavioral assessment of anxiety to detect withdrawal responses after chronic treatment. Diazepam was used as a reference. Mice withdrawn from chronic treatment with diazepam (15 mg/kg/day for 12 days) showed a time-related evolution of anxiety, muscle rigidity and seizures between days 4 and 21 after discontinuation of the treatment. A period between withdrawal days 1 and 3 was symptom free. Mice withdrawn from chronic administration of abecarnil (6 mg/kg/day for 12 days) showed no anxiety and no changes in seizure susceptibility and muscle tone. The doses of diazepam and abecarnil used for chronic treatment were equivalent in terms of kinetics and binding to benzodiazepine receptors. These data indicate that long-term treatment with abecarnil does not induce benzodiazepine-like dependence in mice. Thus, it may be predicted that chronic treatment with abecarnil in humans may offer an important alternative to benzodiazepines in the treatment of anxiety.

Spasticity is characterized by pathological overactivity in spinal stretch reflex circuits and may be associated with disturbances in excitatory amino acid-mediated transmission in the cord. A genetically determined syndrome of spasticity in the rat permits the quantitative evaluation of the antispastic effects of drugs by recording activity in the electromyogram (EMG) from a hind limb extensor muscle. In genetically spastic rats, systemic administration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline (NBQX), normalized pathologically increased EMG activity, whereas the AMPA agonist, alpha-amino-3-hydroxy-5-tertbutyl-4-isoxazolepropionate (ATPA), exacerbated the EMG measures of spasticity. The reflex mechanisms in the spinal cord can be studied in mice using EMG recordings from the tibial muscle (Hoffmann reflex) or from the plantar foot muscle (flexor reflex) after electrical stimulation of the tibial nerve. Systemic and i.t. administration of NBQX blocked Hoffmann reflexes in mice, leaving flexor reflexes unchanged. ATPA enhanced Hoffmann, and had no effect on flexor reflexes. The effects of NBQX on spinal reflexes were seen in doses which do not affect locomotor activity, but show anxiolytic and some antiepileptic activity in rodents. These data suggest that the design of novel muscle relaxant drugs acting at the AMPA subtype of glutamate receptors may be feasible.

Abnormally increased subthalamic nucleus output to the internal pallidal segment and the reticular part of the substantia nigra plays a critical pathophysiological role in the development of parkinsonism. Because synaptic transmission of subthalamic output is glutamatergic and mediated, in part, by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptor, AMPA receptor antagonists may possess antiparkinsonian properties. We report that in monoamine-depleted rats, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) (Novo-Nordisk, Copenhagen, Denmark)--a selective antagonist of the AMPA subtype of glutamate receptor--suppressed muscular rigidity but had no effect on akinesia. NBQX microinjected into the subthalamic nucleus, internal pallidal segment, and reticular part of the substantia nigra, but not into the laterodorsal neostriatum of the rats, stimulated locomotor activity and reduced muscular rigidity. In aged Rhesus monkeys with bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, intramuscular NBQX produced clinically apparent improvement in akinesia, tremor, posture, and gross motor skills. NBQX also potentiated the antiparkinsonian effects of L-3,4-dihydroxyphenylalanine in both rats and monkeys. Blockade of excitatory synaptic transmission by AMPA receptor antagonists may provide a new therapeutic strategy for Parkinson's disease (PD).

Excitatory amino acids are employed as neurotransmitters in the majority of excitatory synapses in the brain (Watkins and Evans, 1981). At present, four subtypes of receptor are recognized, and defined on the basis of their preferred agonists as being N-methyl-D-aspartate (NMDA), quisqualate (Quis), kainate (K) or metabolotropic receptors. Of these, only the NMDA receptor is at all well characterized, largely as a consequence of the availability of specific antagonists acting at a number of apparently different sites on the receptor complex. This receptor gates a channel in neuronal membranes which is permeable to sodium and to calcium. The classical antagonists for NMDA receptors are 2-amino-5-phosphono-alkane carbonic acid derivatives such as 2-amino-5-phosphonopentanoic acid (AP5), which compete with excitatory amino acids for their binding sites at this receptor (e.g. Foster and Fagg, 1984). Additionally, however, a number of well-known pharmacological agents act as non-competitive antagonists, acting at a site apparently within the ion channel; among these are the dissociative anaesthetics such as ketamine (Honey et al., 1985), the hallucinogenic phencyclidine (Anis et al., 1983) and the experimental anticonvulsant, MK 801 (Wong et al., 1986).

Amino acids such as L-glutamate und L-aspartate are major excitatory neurotransmitters in the mammalian central nervous system (CNS) and potential neurotoxins (excitotoxins), which can destroy central neurons by excessive activation of respective receptors. In the last three decades evidence has accumulated that excitatory amino acids (EAA) are involved in many neurological diseases and that pharmacological intervention offers prospects of novel and more effective therapies. Three different receptor types for EAA have been identified, each being named by the selective agonist to which it is preferentially sensitive, i.e. N-methyl-D-aspartate- (NMDA), kainate- and quisqualate-receptors. In this review interest is focused primarily on the NMDA-receptor, whose structure has been subject of numerous electrophysiological and biochemical studies. Today, it is well established that the NMDA-receptor-ionophore complex has an agonist binding site for glutamate, NMDA and related EAAs which is coupled with an ion channel permeable to Na+, K+, Cl- and Ca2+. Four other binding sites for glycine, phencyclidine, Mg2+ and Zn2+ have been identified which can differentially modulate the function of the NMDA receptor. An additional polyamine binding site has recently been reported. Numerous studies on experimental animals demonstrate that modulators of NMDA-mediated neurotransmission may have antiepileptic, anxiolytic, muscle-relaxant and memory-enhancing effects. Particular interest has gained the possible neuroprotective efficacy of NMDA-receptor antagonists in neurological diseases such as hypoxia/ischemia, hypoglycemia, epilepsy and chronic neurodegenerative disorders (Huntington's, Alzheimer's and Parkinson's disease, amyotrophic lateral sclerosis, and AIDS encephalopathy).(ABSTRACT TRUNCATED AT 250 WORDS)

Abecarnil (ZK 112119; isopropyl-6-benzyloxy-4-methoxymethyl-beta-carboxylate) is a metabolically stable beta-carboline derivative with potent anxiolytic and few sedative and ataxic effects in rodents. The anticonvulsant and muscle relaxant actions of abecarnil have been evaluated in mice, rats, gerbils and baboons. Abecarnil raised the threshold for tonic electroconvulsions in mice after corneal but not after auricular application, had no effect on maximal electroshock-induced tonic convulsions triggered by either method, protected mice against the tonic hindlimb extension in PTZ-, picrotoxin- and 3-mercaptopropionate-induced seizures and blocked clonus after PTZ, DMCM (methyl-4-ethyl-6,7-dimethoxy-9H-pyrido-(3,4-b)-indol-3-carboxylate) and 3-mercaptopropionate. Abecarnil had no effect on convulsions induced by bicuculline and strychnine. Furthermore, abecarnil blocked kindled seizures after chronic administration of PTZ and FG 7142 (beta-carboline-3-carboxylic acid methylamide) and protected mice and rats against limbic convulsions induced by pilocarpine. Severity and afterdischarge duration of amygdala-kindled seizures were reduced in rats treated with abecarnil. Abecarnil also antagonized selectively convulsions induced by i.c.v. administration of kainate, but not those triggered by N-methyl-D-aspartate or quisqualate. In genetic models of reflex epilepsy, abecarnil was effective against sound-induced convulsions in DBA/2 mice, against air blast-induced generalized seizures in gerbils and against myoclonus in baboons Papio papio. The anticonvulsant effect of abecornil in a PTZ seizure model in mice was potentiated by ethosuximide, whereas no significant potentiation was found with diazepam, clonazepam, diphenylhydantoin, carbamazepine and phenobarbital. Electromyographic monitoring in a etorphine model of muscle rigidity in rats showed no or little muscle relaxant effect of abecarnil.(ABSTRACT TRUNCATED AT 250 WORDS)

Abecarnil (isopropyl 6-benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate) is a novel ligand for central benzodiazepine (BZ) receptors, possessing anxiolytic and anticonvulsant properties, but with considerably reduced muscle relaxant effects in comparison to diazepam (DZP). In vitro, abecarnil inhibited the binding of the BZ [3H]lormetazepam to rat cerebral cortex membranes with an IC50 value of 0.82 nM in comparison to 56 nM for DZP. The ability of abecarnil to displace [3H]lormetazepam was enhanced 1.24-fold in the presence of 30 microM gamma-aminobutyric acid; the corresponding value for DZP was 2.8-fold. DZP and abecarnil were equally effective in enhancing the binding of t-[35S]butylbicyclophosphorothionate to rat cortical membranes. In vivo, abecarnil exhibited a 3- to 6-fold higher affinity to forebrain BZ receptors than DZP. Abecarnil was from 2 to 10 times more potent than DZP in most rodent tests of anxiolytic activity, and in reducing locomotor activity in mice and rats thoroughly habituated to the test chamber. However, in rats newly exposed to a novel cage, abecarnil was less potent than DZP in reducing locomotor activity. In tests of motor coordination, abecarnil, in contrast to DZP, showed no or only weak activity, and in potentiating the effects of ethanol and hexobarbital on motor performance abecarnil was 4 to 25 times less potent than DZP. Abecarnil antagonized the effects of BZs in the chimney and loss of righting reflex tests, but not in the rotarod test.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonists acting at subtypes of glutamate receptors, N -methyl- d -aspartate, kainate and quisqualate, induce convulsions in rodents. Clonic seizures induced in mice by intracerebral administration of N -methyl- d -aspartate, kainate or quisqualate were used to study the anti- and proconvulsant potential of antiepileptic drugs and β-carbolines. Systemic administration showed that the benzodiazepines clonazepam and midazolam blocked convulsions induced by kainate and had no effect on seizures triggered by N -methyl- d -aspartate and quisqualate. In contrast, diazepam blocked convulsions induced by either excitatory amino acid, as did valproate. The benzodiazepine receptor agonist β-carboline ZK 93423 blocked convulsions induced by kainate but had no effect on seizures induced by N -methyl- d -aspartate or quisqualate. The antagonist β-carboline ZK 93426 did not affect convulsions induced by excitatory amino acids, while the inverse agonists FG 7142 and ethyl-β-carboline-3-carboxylate increased the sensitivity of mice to kainate. Phenobarbital and 2-chloroadenosine protected mice against seizures induced by quisqualate and kainate, while baclofen was active against convulsions produced by kainate. MK-801 selectively blocked convulsions induced by N -methyl- d -aspartate, and enhanced the susceptibility of mice to seizures triggered by kainate and quisqualate. Ethosuximide increased the susceptibility of mice to N -methyl- d -aspartate and had little or no effect on other types of seizures, Diphenylhydantoin enhanced the convulsant potential of quisqualate. Trimethadione and carbamazepine did not affect convulsions induced by N -methyl- d -aspartate, kainate or quisqualate. Intracerebral administration of midazolam protected mice against seizures induced by kainate. Ethosuximide increased the susceptibility of mice to N -methyl- d -aspartate, while diphenylhydantoin to quisqualate convulsions. Phenobarbital and trimethadione had no effect on excitatory amino acid-induced convulsions when injected into the lateral ventricle. The results show that antiepileptic drugs and β-carbolines differentially and selectively affect excitatory neurotransmission mediated by dicarboxylic amino acids.

Systemic administration of morphine hydrochloride (MF; 5-80 mg/kg; i.p.) in rats enhanced the epileptogenic potential of pilocarpine hydrochloride (PIL) in a dose-dependent manner. PIL, 100 mg/kg; i.p., which did not result in convulsions by itself, produced sustained limbic seizures and epileptic brain damage in MF-pretreated rats. MF-induced enhancement of PIL neurotoxicity was blocked by naloxone hydrochloride (NAL; 2 and 10 mg/kg; i.p.). Administration of NAL (2-20 mg/kg) prior to PIL in the dose of 380 mg/kg moderately decreased the incidence of convulsions, brain damage and lethal toxicity produced by this agent. These results support the hypothesis that opiate mechanisms are involved in the maintenance of the threshold for propagation of seizure activity within limbic circuits.

Microinjections of the cholinergic agonists, carbachol and bethanechol, either into the amygdala or into the dorsal hippocampus produced sustained limbic seizures and brain damage in rats. Systemic administration of pilocarpine in rats resulted in a sequence of convulsive disorders and widespread brain damage as well. Scopolamine prevented the development of convulsive activity and brain damage produced by cholinomimetics. These results suggest that the excessive stimulation of cholinergic muscarinic receptors can lead to limbic seizures and brain damage. It is postulated that muscarinic cholinergic mechanisms are linked to the etiology of temporal lobe epilepsy and epileptic brain damage.

Bicuculline methiodide (0.5-3 nmol) and picrotoxin (0.5-4 nmol) were injected uni- or bilaterally into the rat amygdala and the resulting behavioural, electroencephalographic and morphological alterations were studied. In rats treated unilaterally with lowest doses of either bicuculline or picrotoxin (0.5 and 1 nmol) increase in the locomotor activity, occasional myoclonus of the hindlimbs and wet dog shakes were observed. At doses of 2-3 nmol, both gamma-aminobutyrate antagonists produced a sequence of repetitively occurring behavioural alterations including limbic gustatory automatisms, tremor and myoclonus of the forelimbs, head nodding and rearing, that developed over 15-30 min and built up progressively into the recurrent motor limbic seizures lasting for 1-6 h. In animals injected bilaterally with either bicuculline (0.5-3 nmol) or picrotoxin (0.5-3 nmol) motor limbic seizures rapidly developed into the status epilepticus lasting for several hours. Bicuculline and picrotoxin produced both ictal and interictal epileptiform activity in the electroencephalogram. A spectrum of electroencephalographic changes consisted of high voltage fast activity, slow and fast voltage spiking, paraoxysmal bursts and periods of postictal depression. The earliest electrographic alterations appeared in the amygdala and then rapidly spread to cortical areas. Electrographic seizures started 1-10 min after unilateral injections of large doses of bicuculline and pictrotoxin (2-4 nmol). Ictal periods lasted for 1-2 min, recurred every 5-10 min and were followed by periods of depression of the electrographic activity. Bilateral injections of large doses of both gamma-aminobutyrate antagonists (2-3 nmol) resulted in the status epilepticus. Morphological examination of frontal forebrain sections with light microscopy revealed a widespread damage to the amygdala, olfactory cortex, substantia nigra, thalamus, hippocampus and neocortex. Pretreatment of animals with diazepam prevented the build-up of convulsive activity and brain damage produced by bicuculline or picrotoxin. Muscimol retarded the appearance and shortened the duration of convulsive activity, but did not alter the sequence and intensity of seizures. The results indicate that gamma-aminobutyrate antagonists, bicuculline and picrotoxin when directly applied to the amygdala can elicit in rats motor limbic seizures, epileptic changes in the electroencephalogram indicative of repetitive limbic seizures, and status epilepticus accompanied by seizure-related brain damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavioral and neuropathological studies of morphine withdrawal in rats made dependent on the narcotic and precipitated with intracerebral and systemic naloxone or withholding the drug were performed. Unilateral injection of naloxone hydrochloride in the dose of 10 micrograms into the amygdaloid complex elicited severe withdrawal signs including jumping, wet dog shakes, paw tremor, diarrhoea and gustatory automatisms whereas microinjections of naloxone (10 micrograms) into the dorsal hippocampus resulted in severely less pronounced withdrawal behaviors. Histological examination of frontal forebrain sections by light microscopy did not reveal any neuropathological alterations within the brains of rats either dependent on morphine or in those in which morphine withdrawal was precipitated with naloxone or by abrupt termination of morphine intake. The negative finding of the present study does not necessarily mean that there is no relationship between morphine withdrawal and brain damage.

The muscarinic cholinergic agonist pilocarpine induces in rats seizures and status epilepticus followed by widespread damage to the forebrain. The present study was designed to investigate the effect of 5 non-steroidal anti-inflammatory drugs, sodium salicylate, phenylbutazone, indomethacin, ibuprofen and mefenamic acid, on seizures produced by pilocarpine. Pretreatment of rats with sodium salicylate, ED50 103 mg/kg (60-174), and phenylbutazone, 59 mg/kg (50-70) converted the non-convulsant dose of pilocarpine, 200 mg/kg, to a convulsant one. Indomethacin, 1-10 mg/kg, and ibuprofen, 10-100 mg/kg, failed to modulate seizures produced by pilocarpine. Mefenamic acid, 26 (22-30) mg/kg, prevented seizures and protected rats from seizure-related brain damage induced by pilocarpine, 380 mg/kg. These results indicate that non-steroidal anti-inflammatory drugs differentially modulate the threshold for pilocarpine-induced seizures.

Carbachol chloride, administered intracerebroventricularly, induces a characteristic wet dog-shake response in rats. Wet dog shakes induced by carbachol at a dose of 10 μg were antagonized by amitryptyline, imipramine, desipramine, doxepin, clomipramine and maprotyline (tricyclic antidepressants) in a dose-related manner. Iproniazid, nialamide, tranylcypromine and pargyline (monoamine oxidase inhibitors) also effectively antagonized the wet dog-shake response to carbachol. Amphetamine and nomofensine markedly attenuated wet dog shakes, whilst apomorphine failed to inhibit this carbachol-induced response. It is suggested that carbachol-induced shaking behaviour in rats may be a reliable and convenient model for delineating agents with muscarinic anticholinergic activity. In addition, this behavioural syndrome seems to be of particular importance with respect to the pharmacological profile of antidepressant drugs.

The effects of atropine (5 mg/kg), baclofen (10 mg/kg), gamma-hydroxybutyrate (300 mg/kg), gamma-butyrolactone (100 mg/kg) and muscimol (1 mg/kg) upon the action of 2-amino-5-phosphonovalerate (APV; an antagonist at receptors for N-methyl-D-aspartate) on the threshold current for seizures induced by electroshock, were studied in mice. Neither APV, up to 100 mg/kg, nor the other agents produced any significant increase in the convulsive threshold when tested alone. Muscimol had no effect on the action of APV (50 and 100 mg/kg) and the combination of APV with the subthreshold doses of atropine, baclofen, gamma-hydroxybutyrate and gamma-butyrolactone resulted in a clearcut anticonvulsant action. The observed increases in the threshold may be due to the suppressant effects of the drugs upon excitatory transmission, which eventually leads to the potentiation of the action of APV, resulting from blockade of N-methyl-D-aspartate receptors.

2-Amino-5-phosphonopentanoic acid (100 and 200 mg/kg) and 2-amino-7-phosphonoheptanoic acid (50–200 mg/kg i.p.) significantly elevated the threshold for maximal electroconvulsions in mice, the latter being more effective in this respect. In contrast, neither α-aminoadipic acid nor l -glutamic acid diethyl ester (up to 200 and 400 mg/kg, respectively) offered any protection. The present results add further evidence to support the importance of the blockade of N-methyl- d -aspartic acid receptor-mediated events in the suppression of seizure activity.

The effect of aminophylline on the muscle relaxant action of both diazepam and phenobarbitone was studied in genetically spastic rats of the Han-Wistar strain which exhibit spontaneous tonic activity in the electromyogram of the gastrocnemius-soleus muscle. Both diazepam (0.8 and 4.0 mg/kg i.p.) and phenobarbitone (20 and 30 mg/kg i.p.) reduced the spontaneous activity measured in the electromyogram in a dose-related manner. Aminophylline (50 mg/kg i.p.), a methylxanthine with potent antagonistic activity of adenosine-mediated inhibition, partially reversed the muscle relaxant action of diazepam (4 mg/kg) but not that produced by phenobarbitone. The muscle relaxant effect of phenobarbitone (30 mg/kg) was antagonised by beta-carboline-3-carboxylic acid methylester (beta-CCM), 2 mg/kg i.p. The reversal of the muscle relaxant effect of phenobarbitone produced by beta-CCM was abolished by CGS 8216 (2-phenylpyrazolo-(4,3c)quinolin-3(5H)-one), 5 mg/kg i.p. Aminophylline altered neither the muscle relaxant effect of a low dose of diazepam (0.8 mg/kg) nor the reversal of the muscle relaxant effect of phenobarbitone produced by beta-CCM. These findings indicate that the interaction between diazepam and aminophylline does not involve competition for the benzodiazepine receptor and add further support to the suggestion that purinergic mechanisms may be engaged in the muscle relaxant action of diazepam.

The α- and β-stereoisomers of kainate correspond sterically to the l - and d -isomers of glutamate. α-Kainate is a potent excitant at a specific membrane receptor site (kainate receptor). β-Kainate has been proposed as a functional N-methyl- d -aspartate antagonist in vivo . Because of the structural similarities between the α- and β-stereoisomers of kainate we have investigated the interactions of both compounds with N-methyl- d -aspartate-mediated excitation in two well established animal models for assessing the action of excitatory amino acids and their antagonists in vivo : (a) determination of Cd 50 (convulsant dose) for myoclonic seizures in mice and (b) electromyographic measurement of muscle tone in genetically spastic rats. We find that α-kainate and β-kainate produce myoclonic seizures in mice when given intracerebroventricularly and increase the muscle tone in genetically spastic rats when given intrathecally. α-Kainate is about 5000 times more potent than β-kainate as a convulsant and about 1000 times more active than β-kainate in increasing the muscle tone. The excitatory actions of α-kainate and of β -kainate are blocked by γ- d -glutamylaminomethylsulphonate, a preferential kainate/quisqualate antagonist, but not by ( ± )-2-amino-7-phosphonoheptanoate, a specific N-methyl- d -aspartate antagonist. Surprisingly, α-kainate and β-kainate antagonize the myoclonic seizures and the increase in muscle tone produced by N-methyl- d -aspartate and potentiate both the anticonvulsant and myorelaxant actions of ( ± )2-amino-7-phosphonoheptanoate. Quisqualate induces myoclonic seizures in mice after intracerebroventricular application and increases muscle tone in genetically spastic rats following intrathecal injection. Quisqualate potentiates the convulsant action of N-methyl- d -aspartate and does not enhance the anticonvulsant action of ( ± )2-amino-7-phosphonoheptanoateagainst N-methyl- d -aspartate. In genetically spastic rats, quisqualate neither antagonizes the increase in the muscle tone induced by N-methyl- d -aspartate nor enhances the myorelaxant action of ( ± )2-amino-7-phosphonoheptanoate. In conclusion, α-kainate and β-kainate behave in vivo as kainate agonists and as N-methyl- d -aspartate antagonists. Quisqualate does not display N-methyl- d -aspartate antagonist activity in vivo .