Margit Henry

Altered Seizure Susceptibility in Mice Lacking the Cav2.3 E‐type Ca2+ Channel

Summary:  Purpose: Recently the Cav2.3 (E/R‐type) voltage‐gated calcium channel (VGCC) has turned out to be not only a potential target for different antiepileptic drugs (e.g., lamotrigine, topiramate) but also a crucial component in the pathogenesis of absence epilepsy, human juvenile myoclonic epilepsy (JME), and epileptiform activity in CA1 neurons. The aim of our study was to perform an electroencephalographic analysis, seizure‐susceptibility testing, and histomorphologic characterization of Cav2.3−/− mice to unravel the functional relevance of Cav2.3 in ictogenesis.

Design Principles of Concentration-Dependent Transcriptome Deviations in Drug-Exposed Differentiating Stem Cells

Information on design principles governing transcriptome changes upon transition from safe to hazardous drug concentrations or from tolerated to cytotoxic drug levels are important for the application of toxicogenomics data in developmental toxicology. Here, we tested the effect of eight concentrations of valproic acid (VPA; 25–1000 μM) in an assay that recapitulates the development of human embryonic stem cells to neuroectoderm. Cells were exposed to the drug during the entire differentiation process, and the number of differentially regulated genes increased continuously over the concentration range from zero to about 3000. We identified overrepresented transcription factor binding sites (TFBS) as well as superordinate cell biological processes, and we developed a gene ontology (GO) activation profiler, as well as a two-dimensional teratogenicity index. Analysis of the transcriptome data set by the above biostatistical and systems biology approaches yielded the following insights: (i) tolerated (≤25 μM), deregulated/teratogenic (150–550 μM), and cytotoxic (≥800 μM) concentrations could be differentiated. (ii) Biological signatures related to the mode of action of VPA, such as protein acetylation, developmental changes, and cell migration, emerged from the teratogenic concentrations range. (iii) Cytotoxicity was not accompanied by signatures of newly emerging canonical cell death/stress indicators, but by catabolism and decreased expression of cell cycle associated genes. (iv) Most, but not all of the GO groups and TFBS seen at the highest concentrations were already overrepresented at 350–450 μM. (v) The teratogenicity index reflected this behavior, and thus differed strongly from cytotoxicity. Our findings suggest the use of the highest noncytotoxic drug concentration for gene array toxicogenomics studies, as higher concentrations possibly yield wrong information on the mode of action, and lower drug levels result in decreased gene expression changes and thus a reduced power of the study.

Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

The calcium channel α1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of α1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac α1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to α1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of α1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of α1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of α1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of α1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin.

Ablation of Ca(v)2.3 / E-type voltage-gated calcium channel results in cardiac arrhythmia and altered autonomic control within the murine cardiovascular system.

AbstractVoltage–gated calcium channels are key components in cardiac electrophysiology. We demonstrate that Cav2.3 is expressed in mouse and human heart and that mice lacking the Cav2.3 voltage–gated calcium channel exhibit severe alterations in cardiac function. Amplified cDNA fragments from murine heart and single cardiomyocytes reveal the expression of three different Cav2.3 splice variants. The ablation of Cav2.3 was found to be accompanied by a compensatory upregulation of the Cav3.1 T–type calcium channel, while other voltage–gated calcium channels remained unaffected. Telemetric ECG recordings from Cav2.3 deficient mice displayed subsidiary escape rhythm, altered atrial activation patterns, atrioventricular conduction disturbances and alteration in QRS–morphology. Furthermore, time domain analysis of heart rate variability (HRV) in Cav2.3(–|–) mice exhibited a significant increase in heart rate as well as in the coefficient of variance (CV) compared to control mice. Administration of atropin/propranolol revealed that increased heart rate was due to enhanced sympathetic tonus and that partial decrease of CV in Cav2.3(–|–) mice after autonomic block was in accordance with a complete abolishment of 2nd degree atrioventricular block. However, escape rhythms, atrial activation disturbances and QRS–dysmorphology remained unaffected, indicating that these are intrinsic cardiac features in Cav2.3(–|–) mice. We conclude that the expression of Cav2.3 is essential for normal impulse generation and conduction in murine heart.

Immunohistochemical Detection of α1E Voltage-gated Ca2+ Channel Isoforms in Cerebellum, INS-1 Cells, and Neuroendocrine Cells of the Digestive System

Polyclonal antibodies were raised against a common and a specific epitope present only in longer α1E isoforms of voltage-gated Ca2+ channels, yielding an “anti-E-com” and an “anti-E-spec” serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an α1E splice variant with a longer carboxy terminus, the so-called α1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter α1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer α1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, α1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. α1E therefore appears to be a common voltage-gated Ca2+ channel linked to neuroendocrine and related systems of the body. (J Histochem Cytochem 47:981–993, 1999)

Ablation of Cav2.3 / E–type voltage–gatedcalcium channel results in cardiac arrhythmiaand altered autonomic control within themurine cardiovascular system

AbstractVoltage–gated calcium channels are key components in cardiac electrophysiology. We demonstrate that Cav2.3 is expressed in mouse and human heart and that mice lacking the Cav2.3 voltage–gated calcium channel exhibit severe alterations in cardiac function. Amplified cDNA fragments from murine heart and single cardiomyocytes reveal the expression of three different Cav2.3 splice variants. The ablation of Cav2.3 was found to be accompanied by a compensatory upregulation of the Cav3.1 T–type calcium channel, while other voltage–gated calcium channels remained unaffected. Telemetric ECG recordings from Cav2.3 deficient mice displayed subsidiary escape rhythm, altered atrial activation patterns, atrioventricular conduction disturbances and alteration in QRS–morphology. Furthermore, time domain analysis of heart rate variability (HRV) in Cav2.3(–|–) mice exhibited a significant increase in heart rate as well as in the coefficient of variance (CV) compared to control mice. Administration of atropin/propranolol revealed that increased heart rate was due to enhanced sympathetic tonus and that partial decrease of CV in Cav2.3(–|–) mice after autonomic block was in accordance with a complete abolishment of 2nd degree atrioventricular block. However, escape rhythms, atrial activation disturbances and QRS–dysmorphology remained unaffected, indicating that these are intrinsic cardiac features in Cav2.3(–|–) mice. We conclude that the expression of Cav2.3 is essential for normal impulse generation and conduction in murine heart.

Arrhythmia in Isolated Prenatal Hearts after Ablation of the Cav2.3 (α1E) Subunit of Voltage-gated Ca2+ Channels

A voltage-gated calcium channel containing Cav2.3e (α1Ee) as the ion conducting pore has recently been detected in rat heart. Functional evidence for this Ca2+ channel to be involved in the regulation of heart beating, besides L- and T-type channels, was derived from murine embryos where the gene for Cav1.2 had been ablated. The remaining ”L-type like“ current component was not related to recombinant splice variants of Cav1.3 containing channels. As recombinant Cav2.3 channels from rat were reported to be weakly dihydropyridine sensitive, the spontaneous activity of the prenatal hearts from Cav2.3(-|-) mice was compared to that of Cav2.3(+|+) control animals to investigate if Cav2.3 could represent such a L-type like Ca2+ channel. The spontaneous activity of murine embryonic hearts was recorded by using a multielectrode array. Between day 9.5 p.c. to 12.5 p.c., the beating frequency of isolated embryonic hearts from Cav2.3-deficient mice did not differ significantly from control mice but the coefficient of variation within individual episodes was more than four-fold increased in Cav2.3-deficient mice indicating arrhythmia. In isolated hearts from wild type mice, arrhythmia was induced by superfusion with a solution containing 200 nM SNX-482, a blocker of some R-type voltage gated Ca2+ channels, suggesting that R-type channels containing the splice variant Cav2.3e as ion conducting pore stabilize a more regular heart beat in prenatal mice.

Altered thalamocortical rhythmicity in Cav2.3-deficient mice

Voltage-gated calcium channels (VGCCs) are key regulators of neuronal excitability and important factors in epileptogenesis and neurodegeneration. Recent findings suggest a novel, important proictogenic and proneuroapoptotic role of the Ca(v)2.3 E/R-type VGCCs in convulsive generalized tonic-clonic and hippocampal seizures. Though Ca(v)2.3 is also expressed in key structures of the thalamocortical circuitry, their functional relevance in non-convulsive absence seizure activity remains unknown. To this end, we investigated absence specific spike-wave discharge (SWD) susceptibility in control and Ca(v)2.3-deficient mice by systemic administration of gamma-hydroxybutyrolactone (GBL, 70 mg/kg i.p.), followed by electrocorticographic radiotelemetric recordings, behavioral analysis and histomorphological characterization. Based on motoric studies, SWD and power-spectrum density (PSD) analysis, our results demonstrate that Ca(v)2.3(-/-) mice exhibit increased absence seizure susceptibility and altered absence seizure architecture compared to control animals. This study provides evidence for the first time that Ca(v)2.3 E/R-type Ca2+ channels are important in modulating thalamocortical hyperoscillation exerting anti-epileptogenic effects in non-convulsive absence seizures.

Identification of transcriptome signatures and biomarkers specific for potential developmental toxicants inhibiting human neural crest cell migration

The in vitro test battery of the European research consortium ESNATS (‘novel stem cell-based test systems’) has been used to screen for potential human developmental toxicants. As part of this effort, the migration of neural crest (MINC) assay has been used to evaluate chemical effects on neural crest function. It identified some drug-like compounds in addition to known environmental toxicants. The hits included the HSP90 inhibitor geldanamycin, the chemotherapeutic arsenic trioxide, the flame-retardant PBDE-99, the pesticide triadimefon and the histone deacetylase inhibitors valproic acid and trichostatin A. Transcriptome changes triggered by these substances in human neural crest cells were recorded and analysed here to answer three questions: (1) can toxicants be individually identified based on their transcript profile; (2) how can the toxicity pattern reflected by transcript changes be compacted/dimensionality-reduced for practical regulatory use; (3) how can a reduced set of biomarkers be selected for large-scale follow-up? Transcript profiling allowed clear separation of different toxicants and the identification of toxicant types in a blinded test study. We also developed a diagrammatic system to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. activation of KEGG pathways or overrepresentation of gene ontology terms). The transcript data were mined for potential markers of toxicity, and 39 transcripts were selected to either indicate general developmental toxicity or distinguish compounds with different modes-of-action in read-across. In summary, we found inclusion of transcriptome data to largely increase the information from the MINC phenotypic test.

Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two‐step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates.