Publications by authors named "Walter Stühmer"

59 Publications

A new mechanism of voltage-dependent gating exposed by K10.1 channels interrupted between voltage sensor and pore.

J Gen Physiol 2017 May 30;149(5):577-593. Epub 2017 Mar 30.

Oncophysiology Group, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of K10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in K10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism.
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http://dx.doi.org/10.1085/jgp.201611742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412533PMC
May 2017

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel K10.1.

Eur Biophys J 2016 Oct 21;45(7):721-733. Epub 2016 Jul 21.

Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Hermann-Rein-Straße 3, 37075, Göttingen, Germany.

The K10.1 (Eag1) voltage-gated potassium channel represents a promising molecular target for novel cancer therapies or diagnostic purposes. Physiologically, it is only expressed in the brain, but it was found overexpressed in more than 70 % of tumours of diverse origin. Furthermore, as a plasma membrane protein, it is easily accessible to extracellular interventions. In this study we analysed the feasibility of the anti-K10.1 monoclonal antibody mAb62 to target tumour cells in vitro and in vivo and to deliver therapeutics to the tumour. Using time-domain near infrared fluorescence (NIRF) imaging in a subcutaneous MDA-MB-435S tumour model in nude mice, we showed that mAb62-Cy5.5 specifically accumulates at the tumour for at least 1 week in vivo with a maximum intensity at 48 h. Blocking experiments with an excess of unlabelled mAb62 and application of the free Cy5.5 fluorophore demonstrate specific binding to the tumour. Ex vivo NIRF imaging of whole tumours as well as NIRF imaging and microscopy of tumour slices confirmed the accumulation of the mAb62-Cy5.5 in tumours but not in brain tissue. Moreover, mAb62 was conjugated to the prodrug-activating enzyme β-D-galactosidase (β-gal; mAb62-β-gal). The β-gal activity of the mAb62-β-gal conjugate was analysed in vitro on K10.1-expressing MDA-MB-435S cells in comparison to control AsPC-1 cells. We show that the mAb62-β-gal conjugate possesses high β-gal activity when bound to K10.1-expressing MDA-MB-435S cells. Moreover, using the β-gal activatable NIRF probe DDAOG, we detected mAb62-β-gal activity in vivo over the tumour area. In summary, we could show that the anti-K10.1 antibody is a promising tool for the development of novel concepts of targeted cancer therapy.
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http://dx.doi.org/10.1007/s00249-016-1152-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045485PMC
October 2016

Gating Modulation of the Tumor-Related Kv10.1 Channel by Mibefradil.

J Cell Physiol 2017 Aug 11;232(8):2019-2032. Epub 2017 Jan 11.

Max-Planck Institute for Experimental Medicine, Göttingen, Germany.

Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca currents. Given that mibefradil (Mb) is also known to inhibit K channels, we decided to study the interaction between this organic compound and the tumor-related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally, Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. J. Cell. Physiol. 232: 2019-2032, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcp.25448DOI Listing
August 2017

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function.

PLoS One 2016 17;11(3):e0151862. Epub 2016 Mar 17.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET- Partner Institute of the Max Planck Society, Buenos Aires, Argentina.

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151862PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795689PMC
August 2016

SK3 Channel Overexpression in Mice Causes Hippocampal Shrinkage Associated with Cognitive Impairments.

Mol Neurobiol 2017 03 23;54(2):1078-1091. Epub 2016 Jan 23.

Department of Morphology, Physiology and Pathology, CNPQ Research 1B (Biophysics, Biochemistry, Pharmacology and Neuroscience), University of São Paulo Dental School of Ribeirão Preto, Avenida do Café 3400, 14040-904, Ribeirão Preto, Brazil.

The dysfunction of the small-conductance calcium-activated K channel SK3 has been described as one of the factors responsible for the progress of psychoneurological diseases, but the molecular basis of this is largely unknown. This report reveals through use of immunohistochemistry and computational tomography that long-term increased expression of the SK3 small-conductance calcium-activated potassium channel (SK3-T/T) in mice induces a notable bilateral reduction of the hippocampal area (more than 50 %). Histological analysis showed that SK3-T/T mice have cellular disarrangements and neuron discontinuities in the hippocampal formation CA1 and CA3 neuronal layer. SK3 overexpression resulted in cognitive loss as determined by the object recognition test. Electrophysiological examination of hippocampal slices revealed that SK3 channel overexpression induced deficiency of long-term potentiation in hippocampal microcircuits. In association with these results, there were changes at the mRNA levels of some genes involved in Alzheimer's disease and/or linked to schizophrenia, epilepsy, and autism. Taken together, these features suggest that augmenting the function of SK3 ion channel in mice may present a unique opportunity to investigate the neural basis of central nervous system dysfunctions associated with schizophrenia, Alzheimer's disease, or other neuropsychiatric/neurodegenerative disorders in this model system. As a more detailed understanding of the role of the SK3 channel in brain disorders is limited by the lack of specific SK3 antagonists and agonists, the results observed in this study are of significant interest; they suggest a new approach for the development of neuroprotective strategies in neuropsychiatric/neurodegenerative diseases with SK3 representing a potential drug target.
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http://dx.doi.org/10.1007/s12035-015-9680-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310555PMC
March 2017

Support Immersion Endoscopy in Post-Extraction Alveolar Bone Chambers: A New Window for Microscopic Bone Imaging In Vivo.

PLoS One 2015 29;10(12):e0145767. Epub 2015 Dec 29.

Department Oral and Maxillofacial Surgery, Georg-August-University Hospital, Göttingen, Germany.

Using an endoscopic approach, small intraoral bone chambers, which are routinely obtained during tooth extraction and implantation, provide visual in vivo access to internal bone structures. The aim of the present paper is to present a new method to quantify bone microstructure and vascularisation in vivo. Ten extraction sockets and 6 implant sites in 14 patients (6 men / 8 women) were examined by support immersion endoscopy (SIE). After tooth extraction or implant site preparation, microscopic bone analysis (MBA) was performed using short distance SIE video sequences of representative bone areas for off-line analysis with ImageJ. Quantitative assessment of the microstructure and vascularisation of the bone in dental extraction and implant sites in vivo was performed using ImageJ. MBA revealed bone morphology details such as unmineralised and mineralised areas, vascular canals and the presence of bleeding through vascular canals. Morphometric examination revealed that there was more unmineralised bone and less vascular canal area in the implant sites than in the extraction sockets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145767PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695096PMC
July 2016

Analysis of the expression of Kv10.1 potassium channel in patients with brain metastases and glioblastoma multiforme: impact on survival.

BMC Cancer 2015 Nov 3;15:839. Epub 2015 Nov 3.

Department of Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, Goettingen, 37075, Germany.

Background: Kv10.1, a voltage-gated potassium channel only detected in the healthy brain, was found to be aberrantly expressed in extracerebral cancers. Investigations of Kv10.1 in brain metastasis and glioblastoma multiforme (GBM) are lacking.

Methods: We analyzed the expression of Kv10.1 by immunohistochemistry in these brain tumors (75 metastasis from different primary tumors, 71 GBM patients) and the influence of a therapy with tricyclic antidepressants (which are Kv10.1 blockers) on survival. We also investigated Kv10.1 expression in the corresponding primary carcinomas of metastases patients.

Results: We observed positive Kv10.1 expression in 85.3 % of the brain metastases and in 77.5 % of GBMs. Patients with brain metastases, showing low Kv10.1 expression, had a significantly longer overall survival compared to those patients with high Kv10.1 expression. Metastases patients displaying low Kv10.1 expression and also receiving tricyclic antidepressants showed a significantly longer median overall survival as compared to untreated patients.

Conclusions: Our data show that Kv10.1 is not only highly expressed in malignant tumors outside CNS, but also in the most frequent cerebral cancer entities, metastasis and GBM, which remain incurable in spite of aggressive multimodal therapies. Our results extend the correlation between dismal prognosis and Kv10.1 expression to patients with brain metastases or GBMs and, moreover, they strongly suggest a role of tricyclic antidepressants for personalized therapy of brain malignancies.
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http://dx.doi.org/10.1186/s12885-015-1848-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632660PMC
November 2015

Growing neuronal islands on multi-electrode arrays using an accurate positioning-μCP device.

J Neurosci Methods 2016 Jan 1;257:194-203. Epub 2015 Oct 1.

Max Planck Institute for Dynamics and Self-Organization, Dept. Nonlinear Dynamics, Am Faßberg 17, 37077 Göttingen, Germany; Bernstein Center for Computational Neuroscience, Göttingen, Germany; Bernstein Focus Neurotechnology, Göttingen, Germany; SFB-889 Cellular Mechanisms of Sensory Processing, Göttingen, Germany; Faculty of Physics, Georg-August-Universität Göttingen, Göttingen, Germany. Electronic address:

Background: Multi-electrode arrays (MEAs) allow non-invasive multi-unit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEA's surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging.

New Method: In this paper, we present a novel approach utilizing micro-contact printing (μCP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-μCP (accurate positioning micro-contact printing).

Comparison With Existing Methods: Other approaches presented in the literature using μCP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning.

Conclusion: Here we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.
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http://dx.doi.org/10.1016/j.jneumeth.2015.09.022DOI Listing
January 2016

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration.

Neurotox Res 2016 Apr 24;29(3):364-80. Epub 2015 Sep 24.

Sorbonne Université UPMC UM75 INSERM U1127, CNRS UMR 7225, Institut du Cerveau et de la Moelle Epinière, Thérapeutique Expérimentale de la neurodégénérescence, Hôpital de la Salpêtrière - Bâtiment ICM, 47 boulevard de l'Hôpital, 75651, Paris, France.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1(+/+), CX3CR1(+/GFP) and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1(+/GFP) mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.
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http://dx.doi.org/10.1007/s12640-015-9557-5DOI Listing
April 2016

Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator.

Circ Res 2015 Aug 15;117(5):401-12. Epub 2015 Jun 15.

From the Institute of Pharmacology (M.-L.C.L., S.D., E. Wettwer, W.-H.Z.), Clinic for Cardiology and Pulmonology (N.R., E. Wagner, B.U., K.S.-B., K.G., S.W., S.E.L., L.S.M.), and Microarray and Deep-Sequencing Facility (B.R.D.), University Medical Center Göttingen, Göttingen, Germany; DZHK (German Center for Cardiovascular Research), partner site Göttingen, Göttingen, Germany (M.-L.C.L., N.R., E. Wagner, K.S.-B., S.L., K.G., S.E.L., W.S., W.-H.Z.); Institute of Biomechanics, Technical University Hamburg-Harburg, Hamburg, Germany (M.-L.C.L., M.M.M.); Department of Medical Physiology, Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands (T.P.d.B., I.A., T.v.V.); Laboratory of Neuronal Circuit Dynamics, RIKEN Brain Science Institute, Saitama, Japan (H.M., T.K.); Max-Planck-Institutes for Dynamics and Self Organization (N.R., C.R., S.L.) and Experimental Medicine (W.S.), Göttingen, Germany; Department of Internal Medicine II, University Hospital of Regensburg, Regensburg, Germany (B.U., S.W., L.S.M.); Department of Medicine and Centre for Neurotechnology, Imperial College London, United Kingdom (T.K.).

Rationale: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level.

Objective: To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell-derived cardiac myocytes.

Methods And Results: We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell-derived cardiac myocytes.

Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.306143DOI Listing
August 2015

Exocytosis from chromaffin cells: hydrostatic pressure slows vesicle fusion.

Authors:
Walter Stühmer

Philos Trans R Soc Lond B Biol Sci 2015 Jul;370(1672)

Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Hermann-Rein-Strasse 3, Göttingen 37075, Germany

Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å(3) for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes.
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http://dx.doi.org/10.1098/rstb.2014.0192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455761PMC
July 2015

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains.

Nat Commun 2015 Mar 30;6:6672. Epub 2015 Mar 30.

Oncophysiology Group, Max Planck Institute of Experimental Medicine, Hermann-Rein-Straße 3, 37075 Göttingen, Germany.

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.
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http://dx.doi.org/10.1038/ncomms7672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389246PMC
March 2015

KV 10.1 opposes activity-dependent increase in Ca²⁺ influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse.

J Physiol 2015 01 25;593(1):181-96. Epub 2014 Nov 25.

Max-Planck-Institute of Experimental Medicine, 37075, Göttingen, Germany; International Max Planck Research School Neurosciences, 37077, Göttingen, Germany.

Key Points: Voltage-gated KV 10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV 10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV 10.1 regulates Ca(2+) influx and neurotransmitter release during repetitive high-frequency activity. Our results describe the physiological role of mammalian KV 10.1 for the first time and help understand the fine-tuning of synaptic transmission. The voltage-gated potassium channel KV 10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV 10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV 10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca(2+) imaging in KV 10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca(2+) influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre-Purkinje cell synapse at high firing rates. We propose that KV 10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation.
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http://dx.doi.org/10.1113/jphysiol.2014.281600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293062PMC
January 2015

Potassium channels in cell cycle and cell proliferation.

Philos Trans R Soc Lond B Biol Sci 2014 Mar 3;369(1638):20130094. Epub 2014 Feb 3.

Oncophysiology Group, Max Planck Institute of Experimental Medicine, , Hermann-Rein-Strasse 3, Göttingen 37075, Germany.

Normal cell-cycle progression is a crucial task for every multicellular organism, as it determines body size and shape, tissue renewal and senescence, and is also crucial for reproduction. On the other hand, dysregulation of the cell-cycle progression leading to uncontrolled cell proliferation is the hallmark of cancer. Therefore, it is not surprising that it is a tightly regulated process, with multifaceted and very complex control mechanisms. It is now well established that one of those mechanisms relies on ion channels, and in many cases specifically on potassium channels. Here, we summarize the possible mechanisms underlying the importance of potassium channels in cell-cycle control and briefly review some of the identified channels that illustrate the multiple ways in which this group of proteins can influence cell proliferation and modulate cell-cycle progression.
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http://dx.doi.org/10.1098/rstb.2013.0094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917348PMC
March 2014

The roles of K(+) channels in cancer.

Nat Rev Cancer 2014 Jan 12;14(1):39-48. Epub 2013 Dec 12.

Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany.

Potassium channels are transmembrane proteins that selectively facilitate the flow of potassium ions down an electrochemical gradient. These molecules have been studied in great detail in the context of cell excitability, but their roles in less cell type-specific functions, such as cell proliferation, angiogenesis or cell migration, have only recently been assessed. Moreover, the importance of these channels for tumour biology has become evident. This, coupled with the fact that they are accessible proteins and that their pharmacology is well characterized, has increased the interest in investigating potassium channels as therapeutic targets in cancer patients.
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http://dx.doi.org/10.1038/nrc3635DOI Listing
January 2014

KV10.1 K(+)-channel plasma membrane discrete domain partitioning and its functional correlation in neurons.

Biochim Biophys Acta 2014 Mar 22;1838(3):921-31. Epub 2013 Nov 22.

Biochemistry Department, Medicine Faculty, National Autonomous University of Mexico (UNAM), Av. Universidad #3000, 04510 Coyoacán, Mexico City, Mexico. Electronic address:

KV10.1 potassium channels are implicated in a variety of cellular processes including cell proliferation and tumour progression. Their expression in over 70% of human tumours makes them an attractive diagnostic and therapeutic target. Although their physiological role in the central nervous system is not yet fully understood, advances in their precise cell localization will contribute to the understanding of their interactions and function. We have determined the plasma membrane (PM) distribution of the KV10.1 protein in an enriched mouse brain PM fraction and its association with cholesterol- and sphingolipid-rich domains. We show that the KV10.1 channel has two different populations in a 3:2 ratio, one associated to and another excluded from Detergent Resistant Membranes (DRMs). This distribution of KV10.1 in isolated PM is cholesterol- and cytoskeleton-dependent since alteration of those factors changes the relationship to 1:4. In transfected HEK-293 cells with a mutant unable to bind Ca(2+)/CaM to KV10.1 protein, Kv10.1 distribution in DRM/non-DRM is 1:4. Mean current density was doubled in the cholesterol-depleted cells, without any noticeable effects on other parameters. These results demonstrate that recruitment of the KV10.1 channel to the DRM fractions involves its functional regulation.
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http://dx.doi.org/10.1016/j.bbamem.2013.11.007DOI Listing
March 2014

Optogenetic stimulation effectively enhances intrinsically generated network synchrony.

Front Neural Circuits 2013 22;7:167. Epub 2013 Oct 22.

Theoretical Neurophysics, Department of Non-linear Dynamics, Max Planck Institute for Dynamics and Self-Organization Göttingen, Germany ; Max Planck Institute of Experimental Medicine Göttingen, Germany ; Bernstein Focus for Neurotechnology Göttingen, Germany ; Bernstein Center for Computational Neuroscience Göttingen, Germany ; The Interdisciplinary Collaborative Research Center 889 "Cellular Mechanisms of Sensory Processing" Göttingen, Germany.

Synchronized bursting is found in many brain areas and has also been implicated in the pathophysiology of neuropsychiatric disorders such as epilepsy, Parkinson's disease, and schizophrenia. Despite extensive studies of network burst synchronization, it is insufficiently understood how this type of network wide synchronization can be strengthened, reduced, or even abolished. We combined electrical recording using multi-electrode array with optical stimulation of cultured channelrhodopsin-2 transducted hippocampal neurons to study and manipulate network burst synchronization. We found low frequency photo-stimulation protocols that are sufficient to induce potentiation of network bursting, modifying bursting dynamics, and increasing interneuronal synchronization. Surprisingly, slowly fading-in light stimulation, which substantially delayed and reduced light-driven spiking, was at least as effective in reorganizing network dynamics as much stronger pulsed light stimulation. Our study shows that mild stimulation protocols that do not enforce particular activity patterns onto the network can be highly effective inducers of network-level plasticity.
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http://dx.doi.org/10.3389/fncir.2013.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805139PMC
April 2014

Doxycycline restrains glia and confers neuroprotection in a 6-OHDA Parkinson model.

Glia 2013 Jul 17;61(7):1084-100. Epub 2013 Apr 17.

Department of Morphology, Physiology and Pathology, School of Odontology of Ribeirão Preto (FORP), University of São Paulo (USP), Ribeirão Preto, SP, Brazil.

Neuron-glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6-OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6-OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6-OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.
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http://dx.doi.org/10.1002/glia.22496DOI Listing
July 2013

RNA interference with EAG1 enhances interferon gamma injury to glioma cells in vitro.

Anticancer Res 2013 Mar;33(3):865-70

Technology for Gene Therapy Laboratory, University of Brasilia - UnB/FAV, Darcy Ribeiro campus, ICC - ASS 128, Brasília, DF, Brazil.

Aim: The aim of this study was to silence Ether à go-go 1 (EAG1) in glioma cells by RNAi in order to further analyze whether silencing this channel would improve injury caused by interferon gamma (IFN-γ).

Materials And Methods: EAG1 silencing by the siRNAs EAG1hum_287 and EAG1hum_1727 (sequence targets 5'-GGCCTATTGTGTACAGCAATT-3' and 5'-GGGACTTCCTGAAGCTCTATT-3', respectively) was determined by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability was measured by the 3-(4,5)-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. U-138MG glioma cells were injured by IFN-γ (25 ng/ml, 24 h) with or without the RNAi for EAG1 by a non-viral vector (pKV10.1-3, 0.2 μg).

Results: EAG1hum_287 and EAG1hum_1727 caused 0.46- and 0.52-fold decrease in EAG1 mRNA content, respectively. RNAi for EAG1 by pKv10.1-3 strengthened the reduction in cell viability caused by IFN-γ (11.4% versus 40.4%, p<0.05).

Conclusion: The present study reinforces the notion that EAG1 has a role in glioma biology, suggesting that this channel is a relevant player preserving the cell viability during IFN-γ injury.
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March 2013

TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

PLoS One 2012 14;7(12):e51825. Epub 2012 Dec 14.

Department of Molecular Biology of Neuronal Signals, Max-Planck Institute of Experimental Medicine, Göttingen, Germany.

Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051825PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522609PMC
September 2013

Physical and functional interaction of KV10.1 with Rabaptin-5 impacts ion channel trafficking.

FEBS Lett 2012 Sep 25;586(19):3077-84. Epub 2012 Jul 25.

Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute of Experimental Medicine, Göttingen, Germany.

K(V)10.1 is a potassium channel expressed in brain and implicated in tumor progression. We have searched for proteins interacting with K(V)10.1 and identified Rabaptin-5, an effector of the Rab5 GTPase. Both proteins co-localize on large early endosomes induced by Rab5 hyperactivity. Silencing of Rabaptin-5 induces down-regulation of recycling of K(V)10.1 channel in transfected cells and reduction of K(V)10.1 current density in cells natively expressing K(V)10.1, indicating a role of Rabaptin-5 in channel trafficking. K(V)10.1 co-localizes, but does not physically interact, with Rab7 and Rab11. Our data highlights the complex control of the amount of K(V)10.1 channels on the cell surface.
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http://dx.doi.org/10.1016/j.febslet.2012.07.055DOI Listing
September 2012

Rapid internalization of the oncogenic K+ channel K(V)10.1.

PLoS One 2011 12;6(10):e26329. Epub 2011 Oct 12.

Max-Planck-Institute of Experimental Medicine, Department of Molecular Biology of Neuronal Signals, Göttingen, Germany.

K(V)10.1 is a mammalian brain voltage-gated potassium channel whose ectopic expression outside of the brain has been proven relevant for tumor biology. Promotion of cancer cell proliferation by K(V)10.1 depends largely on ion flow, but some oncogenic properties remain in the absence of ion permeation. Additionally, K(V)10.1 surface populations are small compared to large intracellular pools. Control of protein turnover within cells is key to both cellular plasticity and homeostasis, and therefore we set out to analyze how endocytic trafficking participates in controlling K(V)10.1 intracellular distribution and life cycle. To follow plasma membrane K(V)10.1 selectively, we generated a modified channel of displaying an extracellular affinity tag for surface labeling by α-bungarotoxin. This modification only minimally affected K(V)10.1 electrophysiological properties. Using a combination of microscopy and biochemistry techniques, we show that K(V)10.1 is constitutively internalized involving at least two distinct pathways of endocytosis and mainly sorted to lysosomes. This occurs at a relatively fast rate. Simultaneously, recycling seems to contribute to maintain basal K(V)10.1 surface levels. Brief K(V)10.1 surface half-life and rapid lysosomal targeting is a relevant factor to be taken into account for potential drug delivery and targeting strategies directed against K(V)10.1 on tumor cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026329PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192180PMC
February 2012

Tumor cell-selective apoptosis induction through targeting of K(V)10.1 via bifunctional TRAIL antibody.

Mol Cancer 2011 Sep 7;10:109. Epub 2011 Sep 7.

Max-Planck-Institut für Experimentelle Medizin, Hermann-Rein-Str, 3, 37075 Göttingen, Germany.

Background: The search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel K(V)10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins.

Methods: We designed a single-chain antibody against an extracellular region of K(V)10.1 (scFv62) and fused it to the human soluble TRAIL. The K(V)10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity.

Results: Prostate cancer cells, either positive or negative for K(V)10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in K(V)10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking K(V)10.1 expression. In co-cultures with K(V)10.1-positive cancer cells the fusion protein also induced apoptosis in bystander K(V)10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander.

Conclusions: K(V)10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a K(V)10.1-specific antibody.
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http://dx.doi.org/10.1186/1476-4598-10-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179451PMC
September 2011

Eag1 potassium channels as markers of cervical dysplasia.

Oncol Rep 2011 Dec 31;26(6):1377-83. Epub 2011 Aug 31.

Department of Pharmacology, Centro de Investigación y Estudios Avanzados del IPN, Mexico City 07360, Mexico.

Human ether à-go-go 1 (Eag1) potassium channels are potential tumor markers and therapeutic targets for several types of malignancies, including cervical cancer. Estrogens and human papilloma virus oncogenes regulate Eag1 gene expression, suggesting that Eag1 may already be present in pre-malignant lesions. Therefore, Eag1 could be used as an early marker and/or a potential risk indicator for cervical cancer. Consequently, we studied Eag1 protein expression by immunochemistry in cervical cancer cell lines, normal keratinocytes, cervical cytologies from intraepithelial lesions, biopsies from cervical intraepithelial neoplasias (CIN 1, 2 and 3) and in normal smears from patients taking or not taking estrogens. Two hundred and eighty-six samples obtained by liquid-based cytology and fifteen CIN biopsies were studied. We observed Eag1 protein expression in the cervical cancer cell lines, as opposed to normal keratinocytes. Eag1 was found in 67% of the cervical cytologies from low-grade intra-epithelial lesions and in 92% of the samples from high-grade intraepithelial lesions, but only in 27% of the normal samples. Noteworthy, morphologically normal cells obtained from dysplastic samples also exhibited Eag1 expression. In CIN biopsies we found that the higher the grade of the lesion, the broader the Eag1 protein distribution. Almost 50% of the normal patients taking estrogens displayed Eag1 expression. We suggest Eag1 as a potential marker of cervical dysplasia and a risk indicator for developing cervical lesions in patients taking estrogens. Eag1 detection in cervical cancer screening programs should help to improve early diagnosis and decrease mortality rates from this disease.
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http://dx.doi.org/10.3892/or.2011.1441DOI Listing
December 2011

Nucleofection induces non-specific changes in the metabolic activity of transfected cells.

Mol Biol Rep 2012 Mar 5;39(3):2187-94. Epub 2011 Jun 5.

Max-Planck-Institut für experimentelle Medizin, Abteilung Molekulare Biologie Neuronaler Signale, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany.

Transfection has become an everyday technique widely used for functional studies in living cells. The choice of the particular transfection method is usually determined by its efficiency and toxicity, and possible functional consequences specific to the method used are normally overlooked. We describe here that nucleofection, a method increasingly used because of its convenience and high efficiency, increases the metabolic rate of some cancer cells, which can be misleading when used as a measure of proliferation. Moreover, nucleofection can alter the subcellular expression pattern of the transfected protein. These undesired effects are independent of the transfected nucleic acid, but depend on the particular cell line used. Therefore, the interpretation of functional data using this technology requires further controls and caution.
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http://dx.doi.org/10.1007/s11033-011-0967-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271208PMC
March 2012

Functional K(v)10.1 channels localize to the inner nuclear membrane.

PLoS One 2011 May 3;6(5):e19257. Epub 2011 May 3.

Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute of Experimental Medicine, Göttingen, Germany.

Ectopically expressed human K(V)10.1 channels are relevant players in tumor biology. However, their function as ion channels at the plasma membrane does not totally explain their crucial role in tumors. Both in native and heterologous systems, it has been observed that a majority of K(V)10.1 channels remain at intracellular locations. In this study we investigated the localization and possible roles of perinuclear K(V)10.1. We show that K(V)10.1 is expressed at the inner nuclear membrane in both human and rat models; it co-purifies with established inner nuclear membrane markers, shows resistance to detergent extraction and restricted mobility, all of them typical features of proteins at the inner nuclear membrane. K(V)10.1 channels at the inner nuclear membrane are not all transported directly from the ER but rather have been exposed to the extracellular milieu. Patch clamp experiments on nuclei devoid of external nuclear membrane reveal the existence of channel activity compatible with K(V)10.1. We hypothesize that K(V)10.1 channels at the nuclear envelope might participate in the homeostasis of nuclear K(+), or indirectly interact with heterochromatin, both factors known to affect gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019257PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086910PMC
May 2011

A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia.

EMBO Mol Med 2011 Jun 24;3(6):309-19. Epub 2011 Mar 24.

Divison of Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

KCNN3, encoding the small conductance calcium-activated potassium channel SK3, harbours a polymorphic CAG repeat in the amino-terminal coding region with yet unproven function. Hypothesizing that KCNN3 genotypes do not influence susceptibility to schizophrenia but modify its phenotype, we explored their contribution to specific schizophrenic symptoms. Using the Göttingen Research Association for Schizophrenia (GRAS) data collection of schizophrenic patients (n = 1074), we performed a phenotype-based genetic association study (PGAS) of KCNN3. We show that long CAG repeats in the schizophrenic sample are specifically associated with better performance in higher cognitive tasks, comprising the capacity to discriminate, select and execute (p < 0.0001). Long repeats reduce SK3 channel function, as we demonstrate by patch-clamping of transfected HEK293 cells. In contrast, modelling the opposite in mice, i.e. KCNN3 overexpression/channel hyperfunction, leads to selective deficits in higher brain functions comparable to those influenced by SK3 conductance in humans. To conclude, KCNN3 genotypes modify cognitive performance, shown here in a large sample of schizophrenic patients. Reduction of SK3 function may constitute a pharmacological target to improve cognition in schizophrenia and other conditions with cognitive impairment.
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http://dx.doi.org/10.1002/emmm.201100135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377084PMC
June 2011

K(+) channels as therapeutic targets in oncology.

Future Med Chem 2010 May;2(5):745-55

Department of Molecular Biology of Neuronal Signals, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany.

Ion channels are involved in a variety of tumors. In particular, potassium channels are expressed abnormally in many cancer types, where their pharmacologic manipulation impairs tumor progression. Since this group of molecules has been successfully targeted for decades in other therapeutic areas, there is a significant body of knowledge on the pharmacology of potassium channels. Several groups of potassium channels with defined molecular identities have been proposed as candidates for therapeutic intervention. The strategies put forward range from classical small molecule blockade to gene therapy approaches, and include the use of potassium channels as targets for adjuvant therapy. We will discuss the reasons for these proposals and explore possible future developments.
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http://dx.doi.org/10.4155/fmc.10.24DOI Listing
May 2010

Apolipoprotein A-I as a candidate serum marker for the response to lithium treatment in bipolar disorder.

Proteomics 2011 Jan 20;11(2):261-9. Epub 2010 Dec 20.

Institute of Chemistry, University of Campinas (Unicamp), Campinas, Sao Paulo, Brazil.

The molecular basis of bipolar disorder (BD) is still unknown as is the mechanism through which lithium, the therapy of choice, exerts its effects in treatment of BD. So far, no biomarkers exist to facilitate diagnosis of BD or treatment evaluation. To investigate whether BD and its treatment with lithium leaves a characteristic signature in the serum proteome, we used SELDI-TOF MS to analyze individual serum samples from BD patients treated with lithium (BD-plus-Li, n=15) or other drugs (BD-minus-Li, n=10) and from healthy controls (n=15). Interestingly, features of 28 kDa (one peak) and 14 kDa (three peaks) showed a decreased level in the BD-minus-Li group and a level restored to that of the control group in the BD-plus-Li group. To reveal the identity of these features, we subjected pooled serum samples from both BD groups to the 2-D DIGE technology and identified 28 kDa apolipoprotein A-I (apo A-I) and three 14 kDa fragments thereof as upregulated in the BD-plus-Li group. Immunoturbidimetry, a routine clinical assay, verified the characteristic apo A-I signature in individual serum samples. In conclusion, we propose apo A-I as a candidate marker that can visualize response to lithium treatment at the serum protein level.
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http://dx.doi.org/10.1002/pmic.201000371DOI Listing
January 2011

The cross-sectional GRAS sample: a comprehensive phenotypical data collection of schizophrenic patients.

BMC Psychiatry 2010 Nov 10;10:91. Epub 2010 Nov 10.

Division of Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Background: Schizophrenia is the collective term for an exclusively clinically diagnosed, heterogeneous group of mental disorders with still obscure biological roots. Based on the assumption that valuable information about relevant genetic and environmental disease mechanisms can be obtained by association studies on patient cohorts of ≥ 1000 patients, if performed on detailed clinical datasets and quantifiable biological readouts, we generated a new schizophrenia data base, the GRAS (Göttingen Research Association for Schizophrenia) data collection. GRAS is the necessary ground to study genetic causes of the schizophrenic phenotype in a 'phenotype-based genetic association study' (PGAS). This approach is different from and complementary to the genome-wide association studies (GWAS) on schizophrenia.

Methods: For this purpose, 1085 patients were recruited between 2005 and 2010 by an invariable team of traveling investigators in a cross-sectional field study that comprised 23 German psychiatric hospitals. Additionally, chart records and discharge letters of all patients were collected.

Results: The corresponding dataset extracted and presented in form of an overview here, comprises biographic information, disease history, medication including side effects, and results of comprehensive cross-sectional psychopathological, neuropsychological, and neurological examinations. With >3000 data points per schizophrenic subject, this data base of living patients, who are also accessible for follow-up studies, provides a wide-ranging and standardized phenotype characterization of as yet unprecedented detail.

Conclusions: The GRAS data base will serve as prerequisite for PGAS, a novel approach to better understanding 'the schizophrenias' through exploring the contribution of genetic variation to the schizophrenic phenotypes.
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http://dx.doi.org/10.1186/1471-244X-10-91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3002316PMC
November 2010