Hsp90 inhibitor HSP990 in very low dose upregulates EAAT2 and exerts potent antiepileptic activity.
ABSTRACT: Rationale: Dysfunction or reduced levels of EAAT2 have been documented in epilepsy. We previously demonstrated the antiepileptic effects of Hsp90 inhibitor 17AAG in temporal lobe epilepsy by preventing EAAT2 degradation. Because of the potential toxicities of 17AAG, this study aimed to identify an alternative Hsp90 inhibitor with better performance on Hsp90 inhibition, improved blood-brain barrier penetration and minimal toxicity. Methods: We used cell-based screening and animal models of epilepsy, including mouse models of epilepsy and Alzheimer's disease, and a cynomolgus monkey model of epilepsy, to evaluate the antiepileptic effects of new Hsp90 inhibitors. Results: In both primary cultured astrocytes and normal mice, HSP990 enhanced EAAT2 levels at a lower dose than other Hsp90 inhibitors. In epileptic mice, administration of 0.1 mg/kg HSP990 led to upregulation of EAAT2 and inhibition of spontaneous seizures. Additionally, HSP990 inhibited seizures and improved cognitive functions in the APPswe/PS1dE9 transgenic model of Alzheimer's disease. In a cynomolgus monkey model of temporal lobe epilepsy, oral administration of low-dose HSP990 completely suppressed epileptiform discharges for up to 12 months, with no sign of hepatic and renal toxicity. Conclusions: These results support further preclinical studies of HSP990 treatment for temporal lobe epilepsy.
Project description:<h4>Objectives</h4>To study the functional activity of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier of patients with temporal lobe epilepsy using (R)-[(11)C]verapamil (VPM)-PET before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in Pgp function.<h4>Methods</h4>Seven patients with drug-resistant temporal lobe epilepsy underwent VPM-PET scans pre- and postsurgery. Patients were followed up for a median of 6 years (range 4-7) after surgery. Pgp immunoreactivity in surgically resected hippocampal specimens was determined with immunohistochemistry.<h4>Results</h4>Optimal surgical outcome, defined as seizure freedom and withdrawal of antiepileptic drugs, was associated with higher temporal lobe Pgp function before surgery, higher Pgp-positive staining in surgically resected hippocampal specimens, and reduction in global Pgp function postoperatively, compared with nonoptimal surgery outcome.<h4>Conclusions</h4>The data from our pilot study suggest that Pgp overactivity in epilepsy is dynamic, and complete seizure control and elimination of antiepileptic medication is associated with reversal of overactivity, although these findings will require confirmation in a larger patient cohort.
Project description:Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.
Project description:BACKGROUND:The complete assignment of the protein-coding regions of the human genome is a major challenge for genome biology today. We have already isolated many hitherto unknown full-length cDNAs as orthologs of unidentified human genes from cDNA libraries of the cynomolgus monkey (Macaca fascicularis) brain (parietal lobe and cerebellum). In this study, we used cDNA libraries of three other parts of the brain (frontal lobe, temporal lobe and medulla oblongata) to isolate novel full-length cDNAs. RESULTS:The entire sequences of novel cDNAs of the cynomolgus monkey were determined, and the orthologous human cDNA sequences were predicted from the human genome sequence. We predicted 29 novel human genes with putative coding regions sharing an open reading frame with the cynomolgus monkey, and we confirmed the expression of 21 pairs of genes by the reverse transcription-coupled polymerase chain reaction method. The hypothetical proteins were also functionally annotated by computer analysis. CONCLUSIONS:The 29 new genes had not been discovered in recent explorations for novel genes in humans, and the ab initio method failed to predict all exons. Thus, monkey cDNA is a valuable resource for the preparation of a complete human gene catalog, which will facilitate post-genomic studies.
Project description:<h4>Objective</h4>Temporal lobe resection is an established treatment for medication-resistant temporal lobe epilepsy, which in recent years has increasingly been performed in children. However, little is known about the long-term outcome in these children. The aim of this study was to characterize intellectual and psychosocial functioning of children after temporal lobe resection as they progress into late adolescence and adulthood.<h4>Methods</h4>We report the long-term follow-up of 42 children who underwent temporal lobe surgery after an average postoperative period of 9 years. Longitudinal change in IQ was documented, psychosocial outcome including quality of life was assessed, and preoperative and postoperative T1-weighted MRI brain scans were evaluated quantitatively. A well-matched nonsurgical comparison group of 11 children with similar clinical characteristics was also assessed.<h4>Results</h4>At follow-up, 86% of the surgical group were seizure-free, and 57% were no longer taking antiepileptic medication. A significant increase in IQ was found in the surgical group after an extended follow-up period of >5 years. This IQ change was not found in the nonsurgical comparison group. IQ increases were associated with cessation of antiepileptic medication and changes in MRI-derived gray matter volume. The surgical group also reported better psychosocial outcome including quality of life, which was more strongly associated with seizure freedom rather than surgery per se.<h4>Conclusions</h4>Surgery for temporal lobe epilepsy performed in childhood results in excellent long-term seizure control and favorable cognitive outcome along with positive effects on brain development.<h4>Classification of evidence</h4>This study provides Class III evidence that temporal lobectomy in children with temporal lobe epilepsy is associated with improved long-term intellectual outcomes compared with those undergoing standard medical treatment.
Project description:Epilepsy is a common neurological disorder. Despite the availability of a wide range of antiepileptic drugs, these are unsuccessful in preventing seizures in 20-30% of patients. Therefore, new pharmacological strategies are urgently required to control seizures. Modulation of glutamate uptake may have potential in the treatment of pharmacoresistant forms of epilepsy. Previous research showed that the antibiotic ceftriaxone (CTX) increased the expression and functional activity of excitatory amino acid transporter 2 (EAAT2) and exerted considerable anticonvulsant effects. However, other studies did not confirm a significant anticonvulsant effect of CTX administration. We investigated the impacts of CTX treatment on EAAT expression and glutamatergic neurotransmission, as well its anticonvulsant action, in young male Wistar rats. As shown by a quantitative real-time polymerase chain reaction (qPCR) assay and a Western blot analysis, the mRNA but not the protein level of EAAT2 increased in the hippocampus following CTX treatment. Repetitive CTX administration had only a mild anticonvulsant effect on pentylenetetrazol (PTZ)-induced convulsions in a maximal electroshock threshold test (MEST). CTX treatment did not affect the glutamatergic neurotransmission, including synaptic efficacy, short-term facilitation, or the summation of excitatory postsynaptic potentials (EPSPs) in the hippocampus and temporal cortex. However, it decreased the field EPSP (fEPSP) amplitudes evoked by intense electrical stimulation. In conclusion, in young rats, CTX treatment did not induce overexpression of EAAT2, therefore exerting only a weak antiseizure effect. Our data provide new insight into the effects of modulation of EAAT2 expression on brain functioning.
Project description:Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.
Project description:Genetic heterogeneity and signaling alterations diminish the effectiveness of single-agent therapies in glioblastoma multiforme (GBM). HSP90 is a molecular chaperone for several signaling proteins that are deregulated in glioma cells. Thus, HSP90 inhibition may offer an approach to coordinately correct multiple signaling pathways as a strategy for GBM therapy. In this study, we evaluated the effects of a novel HSP90 inhibitor, NVP-HSP990, in glioma tumor-initiating cell (GIC) populations, which are strongly implicated in the root pathobiology of GBM. In GIC cultures, NVP-HSP990 elicited a dose-dependent growth inhibition with IC50 values in the low nanomolar range. Two GIC subgroups with different responses were observed with an Olig2-expressing subset relatively more sensitive to treatment. We also showed that Olig2 is a functional marker associated with cell proliferation and response to NVP-HSP990, as NVP-HSP990 attenuated cell proliferation in Olig2-high GIC lines. In addition, NVP-HSP990 disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and Olig2. Inhibition of CDK2/CDK4 activity disrupted Olig2-CDK2/CDK4 interactions and attenuated Olig2 protein stability. In vivo evaluation showed a relative prolongation of median survival in an intracranial model of GIC growth. Our results suggest that GBM characterized by high-expressing Olig2 GIC may exhibit greater sensitivity to NVP-HSP990 treatment, establishing a foundation for further investigation of the role of HSP90 signaling in GBM.
Project description:C-X-C motif chemokine receptor 7 (CXCR7), which mediates the immune response in the brain, was recently reported to regulate neurological functions. However, the role of CXCR7 in epilepsy remains unclear. Here, we found that CXCR7 was upregulated in the hippocampal dentate gyrus (DG) of mice subjected to kainic acid (KA)-induced epilepsy and in the brain tissues of patients with temporal lobe epilepsy. Silencing CXCR7 in the hippocampal DG region exerted an antiepileptic effect on the KA-induced mouse model of epilepsy, whereas CXCR7 overexpression produced a seizure-aggravating effect. Mechanistically, CXCR7 selectively regulated N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic neurotransmission in hippocampal dentate granule cells by modulating the cell membrane expression of the NMDAR subunit2A, which requires the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Thus, CXCR7 may regulate epileptic seizures and represents a novel target for antiepileptic treatments.
Project description:Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH?). The formation of 17AAGH? by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH? were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH? could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH?), reduction of 17AAG to 17AAGH? by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH?. In addition, oxidation of 17AAGH? could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH? was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH? with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH?, the active Hsp90 inhibitor derived from 17AAG.
Project description:Antiepileptic drugs impair episodic memory in patients with epilepsy, but this effect has so far only been examined with tests that do not provide first-person experience-an aspect that is crucial for episodic memory. Virtual reality techniques facilitate the development of ecologically valid tests. In the present study, we measure the effect of antiepileptic drug changes in a within-subject design using a virtual reality test in order to provide direct evidence for effects of antiepileptic drugs on episodic memory. Among 106 recruited patients, 97 participated in a virtual reality test up to six times during a 4-day hospitalization, and 78 patients underwent changes in drug load during this period. There were six parallel versions of a virtual town test, with immediate recall and delayed recall after about 12 h. The test requires recall of elements, details, sequence of experience, and egocentric and allocentric spatial memory. We determined drug load by defined daily dose, and compared test performance at lowest antiepileptic drug load to highest antiepileptic drug load. Across the six towns, performance was lower in delayed compared to immediate recall. There was an overall effect of medication when comparing patients taking vs. not taking antiepileptic drugs and/or psychoactive drugs (p = 0.005). Furthermore, there was a within-subject effect of antiepileptic drug load (p = 0.01), indicating lower test performance at higher drug load. There was no effect of gender, daytime, circadian type, depression, seizures, lesions, and epilepsy. For patients with temporal lobe epilepsy, there was no effect of lateralization. The present study provides direct evidence for episodic memory impairment due to antiepileptic drugs, suggesting that a small change in drug load can matter. This study can serve as a proof of principle for the methodology, but a larger sample is needed to examine the differential effects of individual antiepileptic drugs.