Christina Schaub

Transcriptional Regulation of T-type Calcium Channel CaV3.2 BI-DIRECTIONALITY BY EARLY GROWTH RESPONSE 1 (Egr1) AND REPRESSOR ELEMENT 1 (RE-1) PROTEIN -SILENCING TRANSCRIPTION FACTOR (REST)

Background: Expression of the T-type Ca2+-channel CaV3.2 has to be tightly regulated for proper calcium homeostasis. Results: Overexpression of the transcription factor Egr1 strongly activates the CaV3.2 promoter and can be counteracted by the repressor REST. Conclusion: Egr1 and REST “bi-directionally” regulate the CaV3.2 promoter. Significance: Our results have important implications for calcium homeostasis and dynamics in health and disease. The pore-forming Ca2+ channel subunit CaV3.2 mediates a low voltage-activated (T-type) Ca2+ current (ICaT) that contributes pivotally to neuronal and cardiac pacemaker activity. Despite the importance of tightly regulated CaV3.2 levels, the mechanisms regulating its transcriptional dynamics are not well understood. Here, we have identified two key factors that up- and down-regulate the expression of the gene encoding CaV3.2 (Cacna1h). First, we determined the promoter region and observed several stimulatory and inhibitory clusters. Furthermore, we found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the CaV3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the CaV3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo. Subsequent chromatin immunoprecipitation assays in NG108-15 cells and mouse hippocampi confirmed specific Egr1 binding to the CaV3.2 promoter. Congruently, whole-cell ICaT values were significantly larger after Egr1 overexpression. Intriguingly, Egr1-induced activation of the CaV3.2 promoter was effectively counteracted by the repressor element 1-silencing transcription factor (REST). Thus, Egr1 and REST can bi-directionally regulate CaV3.2 promoter activity and mRNA expression and, hence, the size of ICaT. This mechanism has critical implications for the regulation of neuronal and cardiac Ca2+ homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy.

Targeting pharmacoresistant epilepsy and epileptogenesis with a dual-purpose antiepileptic drug

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting a substantial fraction of patients. Many of the currently available antiepileptic drugs target voltage-gated sodium channels, leading to a rate-dependent suppression of neuronal discharge. A loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. There is a need both for compounds that overcome this resistance mechanism and for novel drugs that inhibit the process of epileptogenesis. We show that eslicarbazepine acetate, a once-daily antiepileptic drug, may constitute a candidate compound that addresses both issues. Eslicarbazepine acetate is converted extensively to eslicarbazepine after oral administration. We have first tested using patch-clamp recording in human and rat hippocampal slices if eslicarbazepine, the major active metabolite of eslicarbazepine acetate, shows maintained activity in chronically epileptic tissue. We show that eslicarbazepine exhibits maintained use-dependent blocking effects both in human and experimental epilepsy with significant add-on effects to carbamazepine in human epilepsy. Second, we show that eslicarbazepine acetate also inhibits Cav3.2 T-type Ca(2+) channels, which have been shown to be key mediators of epileptogenesis. We then examined if transitory administration of eslicarbazepine acetate (once daily for 6 weeks, 150 mg/kg or 300 mg/kg) after induction of epilepsy in mice has an effect on the development of chronic seizures and neuropathological correlates of chronic epilepsy. We found that eslicarbazepine acetate exhibits strong antiepileptogenic effects in experimental epilepsy. EEG monitoring showed that transitory eslicarbazepine acetate treatment resulted in a significant decrease in seizure activity at the chronic state, 8 weeks after the end of treatment. Moreover, eslicarbazepine acetate treatment resulted in a significant decrease in mossy fibre sprouting into the inner molecular layer of pilocarpine-injected mice, as detected by Timm staining. In addition, epileptic animals treated with 150 mg/kg, but not those that received 300 mg/kg eslicarbazepine acetate showed an attenuated neuronal loss. These results indicate that eslicarbazepine potentially overcomes a cellular resistance mechanism to conventional antiepileptic drugs and at the same time constitutes a potent antiepileptogenic agent.

Diminished Response of CA1 Neurons to Antiepileptic Drugs in Chronic Epilepsy

Summary  Purpose: A substantial proportion of epilepsy patients (∼30%) continue to have seizures despite carefully optimized treatment with antiepileptic drugs (AEDs). One key concept to explain the development of pharmacoresistance is that epilepsy‐related changes in the properties of CNS drug targets result in AED‐insensitivity of these targets. These changes then contribute to drug‐resistance on a clinical level. We have tested this hypothesis in hippocampal CA1 neurons in experimental epilepsy.

T-type Ca2+ channels encode prior neuronal activity as modulated recovery rates.

T‐type Ca2+ channels give rise to low‐threshold inward currents that are central determinants of neuronal excitability. The availability of T‐type Ca2+ channels is strongly influenced by voltage‐dependent inactivation and recovery from inactivation. Here, we show that native and cloned T‐type Ca2+ channel subunits selectively encode specific aspects of prior membrane potential changes via a powerful modulation of the rates with which these channels recover from inactivation. Increasing the duration of subthreshold (−70 to −55 mV) conditioning depolarizations caused a pronounced slowing of subsequent recovery from inactivation of both cloned (Cav3.1–3.3) and native T‐type channels (thalamic neurones). The scaling of recovery rates with increasing duration of conditioning depolarizations could be well described by a power law function. Different T‐type channel isoforms exhibited overlapping but complementary ranges of recovery rates. Intriguingly, scaling of recovery rates was dramatically reduced in Cav3.2 and Cav3.3, but not Cav3.1 subunits, when mock action potentials were superimposed on conditioning depolarizations. Our results suggest that different T‐type channel subunits exhibit dramatic differences in scaling relationships, in addition to well‐described differences in other biophysical properties. Furthermore, the availability of T‐type channels is powerfully modulated over time, depending on the patterns of prior activity that these channels have encountered. These data provide a novel mechanism for cellular short‐term plasticity on the millisecond to second time scale that relies on biophysical properties of specific T‐type Ca2+ channel subunits.

Zinc regulates a key transcriptional pathway for epileptogenesis via metal-regulatory transcription factor 1

Temporal lobe epilepsy (TLE) is the most common focal seizure disorder in adults. In many patients, transient brain insults, including status epilepticus (SE), are followed by a latent period of epileptogenesis, preceding the emergence of clinical seizures. In experimental animals, transcriptional upregulation of CaV3.2 T-type Ca2+-channels, resulting in an increased propensity for burst discharges of hippocampal neurons, is an important trigger for epileptogenesis. Here we provide evidence that the metal-regulatory transcription factor 1 (MTF1) mediates the increase of CaV3.2 mRNA and intrinsic excitability consequent to a rise in intracellular Zn2+ that is associated with SE. Adeno-associated viral (rAAV) transfer of MTF1 into murine hippocampi leads to increased CaV3.2 mRNA. Conversely, rAAV-mediated expression of a dominant-negative MTF1 abolishes SE-induced CaV3.2 mRNA upregulation and attenuates epileptogenesis. Finally, data from resected human hippocampi surgically treated for pharmacoresistant TLE support the Zn2+-MTF1-CaV3.2 cascade, thus providing new vistas for preventing and treating TLE.

Carmustine (BCNU) plus Teniposide (VM26) in Recurrent Malignant Glioma

Background: After the failure of radiotherapy and temozolomide, there is no established standard therapy for patients with recurrent glioblastoma (GBM). Based on the promising data of a previous trial (NOA-01) for primary GBM and some retrospective case series for GBM recurrence, the combination of nimustine and teniposide (VM26) was commonly used in this setting. When nimustine was no longer available in Europe, we switched to intrvaveneous carmustine (BCNU). Data on the toxicity and efficacy of BCNU and VM26 in recurrent GBM are lacking. Methods: In our neurooncological center, all patients with recurrent GBM or with progressed glioma and a typical MRI lesion suggesting GBM treated with BCNU (130-150 mg/m2, day 1/42) and VM26 (45-60 mg/m2, days 1-3/42) were analyzed retrospectively for progression-free survival, overall survival and toxicity. Results: Fifteen patients (median age 52 years) were identified. Median progression-free survival was 2 months and median overall survival was 4 months. Two patients (14%) developed grade 3/4 hematotoxicity. Nonhematological toxicity ≥grade 3 was not observed. Conclusion: Our data do not support the application of BCNU/VM26 in patients with late stages of recurrent GBM.

Quality of life in the GLARIUS trial randomizing bevacizumab/irinotecan versus temozolomide in newly diagnosed, MGMT-nonmethylated glioblastoma

Background The GLARIUS trial, which investigated the efficacy of bevacizumab (BEV)/irinotecan (IRI) compared with standard temozolomide in the first-line therapy of O6-methylguanine-DNA methyltransferase (MGMT)-nonmethylated glioblastoma, showed that progression-free survival was significantly prolonged by BEV/IRI, while overall survival was similar in both arms. The present report focuses on quality of life (QoL) and Karnofsky performance score (KPS) during the whole course of the disease. Methods Patients (n = 170) received standard radiotherapy and were randomized (2:1) for BEV/IRI or standard temozolomide. At least every 3 months KPS was determined and QoL was measured using the European Organisation for Research and Treatment of Cancer 30-item Core Quality of Life and 20-item Brain Neoplasm questionnaires. A generalized estimating equation (GEE) model evaluated differences in the course of QoL and KPS over time. Also, the time to first deterioration and the time to postprogression deterioration were analyzed separately. Results In all dimensions of QoL and KPS, GEE analyses and time to first deterioration analyses did not detect significant differences between the treatment arms. At progression, 82% of patients receiving second-line therapy in the standard arm received BEV second-line therapy. For the dimensions of motor dysfunction and headaches, time to postprogression deterioration was prolonged in the standard arm receiving crossover second-line BEV in the vast majority of patients at the time of evaluation. Conclusions GLARIUS did not find indications for a BEV-induced detrimental effect on QoL in first-line therapy of MGMT-nonmethylated GBM patients. Moreover, GLARIUS provided some indirect corroborative data supporting the notion that BEV may have beneficial effects upon QoL in relapsed GBM.

Tumor growth patterns of MGMT-non-methylated glioblastoma in the randomized GLARIUS trial

BackgroundWe evaluated patterns of tumor growth in patients with newly diagnosed MGMT-non-methylated glioblastoma who were assigned to undergo radiotherapy in conjunction with bevacizumab/irinotecan (BEV/IRI) or standard temozolomide (TMZ) within the randomized phase II GLARIUS trial.MethodsIn 142 patients (94 BEV/IRI, 48 TMZ), we reviewed magnetic resonance imaging scans at baseline and first tumor recurrence. Based on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery images, we assessed tumor growth patterns and tumor invasiveness. Tumor growth patterns were classified as either multifocal or local at baseline and recurrence; at first recurrence, we additionally assessed whether distant lesions appeared. Invasiveness was determined as either diffuse or non-diffuse. Associations with treatment arms were calculated using Fisher’s exact test.ResultsAt baseline, 115 of 142 evaluable patients (81%) had a locally confined tumor. Between treatment arms, there was no significant difference in the fraction of tumors that changed from an initially local tumor growth pattern to a multifocal pattern (12 and 13%, p = 0.55). Distant lesions appeared in 17% (BEV/IRI) and 13% (TMZ) of patients (p = 0.69). 15% of patients in the BEV/IRI arm and 8% in the TMZ arm developed a diffuse growth pattern from an initially non-diffuse pattern (p = 0.42).ConclusionsThe tumor growth and invasiveness patterns do not differ between BEV/IRI and TMZ-treated MGMT-non-methylated glioblastoma patients in the GLARIUS trial. BEV/IRI was not associated with an increased rate of multifocal, distant, or highly invasive tumors at the time of recurrence.