Publications by authors named "Christoph Schwarzer"

67 Publications

Knockout of Ca1.3 L-type calcium channels in a mouse model of retinitis pigmentosa.

Sci Rep 2021 Jul 26;11(1):15146. Epub 2021 Jul 26.

Institute of Pharmacy, Pharmacology and Toxicology, Center for Chemistry and Biomedicine, University of Innsbruck, Innrain 80-82, 6020, Innsbruck, Austria.

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.
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http://dx.doi.org/10.1038/s41598-021-94304-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313562PMC
July 2021

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Handb Exp Pharmacol 2021 Jun 4. Epub 2021 Jun 4.

Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.
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http://dx.doi.org/10.1007/164_2021_467DOI Listing
June 2021

The Kappa Opioid Receptor System in Temporal Lobe Epilepsy.

Handb Exp Pharmacol 2021 Mar 13. Epub 2021 Mar 13.

Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Temporal lobe epilepsy is considered to be one of the most common and severe forms of focal epilepsies. Patients frequently develop cognitive deficits and emotional blunting along progression of the disease. The high incidence of refractoriness to antiepileptic drugs and a frequent lack of admissibility to surgery pose an unmet medical challenge. In the urgent quest for novel treatment strategies, neuropeptides and their receptors are interesting candidates. However, their therapeutic potential has not yet been fully exploited. This chapter focuses on the functional role of the dynorphins (Dyns) and the kappa opioid receptor (KOR) system in temporal lobe epilepsy and the hippocampus.Genetic polymorphisms in the prepro-dynorphin (pDyn) gene causing lower levels of Dyns in humans and pDyn gene knockout in mice increase the risk to develop epilepsy. This suggests a role of Dyns and KOR as modulators of neuronal excitability. Indeed, KOR agonists induce inhibition of presynaptic neurotransmitter release, as well as postsynaptic hyperpolarization in glutamatergic neurons, both producing anticonvulsant effects.The development of new approaches to modulate the complex KOR signalling cascade (e.g. biased agonism and gene therapy) opens up new exciting therapeutic opportunities with regard to seizure control and epilepsy. Potential adverse side effects of KOR agonists may be minimized through functional selectivity or locally restricted treatment. Preclinical data suggest a high potential of such approaches to control seizures.
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http://dx.doi.org/10.1007/164_2021_444DOI Listing
March 2021

Phenotypic Characterization and Brain Structure Analysis of Calcium Channel Subunit αδ-2 Mutant (Ducky) and αδ Double Knockout Mice.

Front Synaptic Neurosci 2021 19;13:634412. Epub 2021 Feb 19.

Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria.

Auxiliary αδ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (αδ-1, αδ-2, and αδ-3) are abundantly expressed in the brain; however, of the available knockout models, only αδ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal αδ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct αδ double knockout mouse models by crossbreeding single knockout (αδ-1 and -3) or mutant (αδ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct αδ-1/-2, αδ-1/-3, and αδ-2/-3 breeding combinations did not conform to Mendel's law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific αδ isoforms (αΔ-2 > αδ-1 > αδ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of αδ-2/-3 cross-breedings. Juvenile αδ-1/-2 and αδ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and αδ-1/-3 double knockout animals, αδ-1/-2 and αδ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that αδ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.
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http://dx.doi.org/10.3389/fnsyn.2021.634412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933509PMC
February 2021

A Rationale for Hypoxic and Chemical Conditioning in Huntington's Disease.

Int J Mol Sci 2021 Jan 8;22(2). Epub 2021 Jan 8.

Institute of Virology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 12203 Berlin, Germany.

Neurodegenerative diseases are characterized by adverse cellular environments and pathological alterations causing neurodegeneration in distinct brain regions. This development is triggered or facilitated by conditions such as hypoxia, ischemia or inflammation and is associated with disruptions of fundamental cellular functions, including metabolic and ion homeostasis. Targeting intracellular downstream consequences to specifically reverse these pathological changes proved difficult to translate to clinical settings. Here, we discuss the potential of more holistic approaches with the purpose to re-establish a healthy cellular environment and to promote cellular resilience. We review the involvement of important molecular pathways (e.g., the sphingosine, δ-opioid receptor or N-Methyl-D-aspartate (NMDA) receptor pathways) in neuroprotective hypoxic conditioning effects and how these pathways can be targeted for chemical conditioning. Despite the present scarcity of knowledge on the efficacy of such approaches in neurodegeneration, the specific characteristics of Huntington's disease may make it particularly amenable for such conditioning techniques. Not only do classical features of neurodegenerative diseases like mitochondrial dysfunction, oxidative stress and inflammation support this assumption, but also specific Huntington's disease characteristics: a relatively young age of neurodegeneration, molecular overlap of related pathologies with hypoxic adaptations and sensitivity to brain hypoxia. The aim of this review is to discuss several molecular pathways in relation to hypoxic adaptations that have potential as drug targets in neurodegenerative diseases. We will extract the relevance for Huntington's disease from this knowledge base.
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http://dx.doi.org/10.3390/ijms22020582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826574PMC
January 2021

Biallelic mutations in the death domain of PIDD1 impair caspase-2 activation and are associated with intellectual disability.

Transl Psychiatry 2021 01 5;11(1). Epub 2021 Jan 5.

Molecular Neuropsychiatry & Development (MiND) Lab, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8, Canada.

PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD-the protein bridging PIDD1 and caspase-2-have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1, all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.
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http://dx.doi.org/10.1038/s41398-020-01158-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791037PMC
January 2021

Mitochondrial Respiration Changes in R6/2 Huntington's Disease Model Mice during Aging in a Brain Region Specific Manner.

Int J Mol Sci 2020 Jul 30;21(15). Epub 2020 Jul 30.

Huntington and Rare Diseases Unit, Fondazione IRCCS Casa Sollievo della Sofferenza Research Hospital, 71013 San Giovanni Rotondo, Italy.

Mitochondrial dysfunction is crucially involved in aging and neurodegenerative diseases, such as Huntington's Disease (HD). How mitochondria become compromised in HD is poorly understood but instrumental for the development of treatments to prevent or reverse resulting deficits. In this paper, we investigate whether oxidative phosphorylation (OXPHOS) differs across brain regions in juvenile as compared to adult mice and whether such developmental changes might be compromised in the R6/2 mouse model of HD. We study OXPHOS in the striatum, hippocampus, and motor cortex by high resolution respirometry in female wild-type and R6/2 mice of ages corresponding to pre-symptomatic and symptomatic R6/2 mice. We observe a developmental shift in OXPHOS-control parameters that was similar in R6/2 mice, except for cortical succinate-driven respiration. While the LEAK state relative to maximal respiratory capacity was reduced in adult mice in all analyzed brain regions, succinate-driven respiration was reduced only in the striatum and cortex, and NADH-driven respiration was higher as compared to juvenile mice only in the striatum. We demonstrate age-related changes in respirational capacities of different brain regions with subtle deviations in R6/2 mice. Uncovering in situ oxygen conditions and potential substrate limitations during aging and HD disease progression are interesting avenues for future research to understand brain-regional vulnerability in HD.
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http://dx.doi.org/10.3390/ijms21155412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432063PMC
July 2020

Neuroinflammatory alterations in trait anxiety: modulatory effects of minocycline.

Transl Psychiatry 2020 07 30;10(1):256. Epub 2020 Jul 30.

Department of Pharmacology and Toxicology, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.

High trait anxiety is a substantial risk factor for developing anxiety disorders and depression. While neuroinflammation has been identified to contribute to stress-induced anxiety, little is known about potential dysregulation in the neuroinflammatory system of genetically determined pathological anxiety or high trait anxiety individuals. We report microglial alterations in various brain regions in a mouse model of high trait anxiety (HAB). In particular, the dentate gyrus (DG) of the hippocampus of HABs exhibited enhanced density and average cell area of Iba1+, and density of phagocytic (CD68+/Iba1+) microglia compared to normal anxiety (NAB) controls. Minocycline was used to assess the capacity of a putative microglia 'inhibitor' in modulating hyperanxiety behavior of HABs. Chronic oral minocycline indeed reduced HAB hyperanxiety, which was associated with significant decreases in Iba1+ and CD68+Iba1+ cell densities in the DG. Addressing causality, it was demonstrated that longer (10 days), but not shorter (5 days), periods of minocycline microinfusions locally into the DG of HAB reduced Iba-1+ cell density and attenuated hyperanxiety-related behavior, indicating that neuroinflammation in the DG is at least partially involved in the maintenance of pathological anxiety. The present data reveal evidence of disturbances in the microglial system of individuals with high trait anxiety. Minocycline attenuated HAB hyperanxiety, likely by modulation of microglial activity within the DG. Thus, the present data suggest that drugs with microglia-targeted anti-inflammatory properties could be promising as novel alternative or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals genetically predisposed to hyperanxiety.
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http://dx.doi.org/10.1038/s41398-020-00942-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393101PMC
July 2020

Impaired chloride homeostasis in epilepsy: Molecular basis, impact on treatment, and current treatment approaches.

Pharmacol Ther 2020 01 15;205:107422. Epub 2019 Oct 15.

Department of Pharmacology, Medical University of Innsbruck, Peter-Mayr-Str. 1a, 6020 Innsbruck, Austria. Electronic address:

Epilepsies represent one of the most common neurological diseases worldwide. They are characterized by recurrent spontaneous seizures with severe impact on a patient's life. An imbalance in excitatory and inhibitory signalling is considered the main underlying pathophysiological mechanism. Therefore, GABA-mimetic drugs, strengthening the main inhibitory signalling system in the CNS, are frequently used as antiepileptic or anticonvulsant drugs. However, the therapeutic effect of such treatment depends on the chloride gradient along the plasma membrane. Impairment of chloride homeostasis, caused by alterations in the functional balance of chloride transporters, was implicated in the pathophysiology of epilepsy and numerous other diseases. Breakdown or even inversion of the chloride gradient may result in ineffective or in worst cases proconvulsant effects of GABA-mimetics. Unfortunately, such situations are reported in considerable number. Consequently, bumetanide, an inhibitor of Na-K-Cl cotransporters gained interest as potential add-on therapy re-establishing the chloride gradient and thereby the hyperpolarizing effects of GABA-mimetic drugs. Indeed, preclinical studies yielded encouraging results, especially when applied in combination with GABA-mimetics in epilepsy models. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier, two adverse features for chronic antiepileptic treatment. Therefore, new compounds overcoming these limitations are under development. This review focuses on alterations in chloride homeostasis and its underlying molecular mechanisms in epilepsy, on the potential impact of impaired chloride homeostasis on the treatment of epilepsy and on concepts to overcome this problem including recent development of bumetanide derivatives with improved pharmacological profile.
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http://dx.doi.org/10.1016/j.pharmthera.2019.107422DOI Listing
January 2020

Dynorphin-based "release on demand" gene therapy for drug-resistant temporal lobe epilepsy.

EMBO Mol Med 2019 10 5;11(10):e9963. Epub 2019 Sep 5.

Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Focal epilepsy represents one of the most common chronic CNS diseases. The high incidence of drug resistance, devastating comorbidities, and insufficient responsiveness to surgery pose unmet medical challenges. In the quest of novel, disease-modifying treatment strategies of neuropeptides represent promising candidates. Here, we provide the "proof of concept" that gene therapy by adeno-associated virus (AAV) vector transduction of preprodynorphin into the epileptogenic focus of well-accepted mouse and rat models for temporal lobe epilepsy leads to suppression of seizures over months. The debilitating long-term decline of spatial learning and memory is prevented. In human hippocampal slices obtained from epilepsy surgery, dynorphins suppressed seizure-like activity, suggestive of a high potential for clinical translation. AAV-delivered preprodynorphin expression is focally and neuronally restricted and release is dependent on high-frequency stimulation, as it occurs at the onset of seizures. The novel format of "release on demand" dynorphin delivery is viewed as a key to prevent habituation and to minimize the risk of adverse effects, leading to long-term suppression of seizures and of their devastating sequel.
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http://dx.doi.org/10.15252/emmm.201809963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783645PMC
October 2019

Functional characterization of novel bumetanide derivatives for epilepsy treatment.

Neuropharmacology 2020 01 30;162:107754. Epub 2019 Aug 30.

Department of Pharmacology, Medical University of Innsbruck, Peter-Mayr-Str. 1a, 6020, Innsbruck, Austria. Electronic address:

Temporal lobe epilepsy (TLE) is the most common type of focal epilepsies, affecting approximately 35 million people worldwide. Despite the introduction of numerous novel antiepileptic drugs during the last decades, the proportion of patients with therapy-resistant TLE is still high. As an impaired cellular chloride homeostasis appears involved in disease pathophysiology, bumetanide, an antagonist to Na-K-Cl cotransporters, gained interest as potential therapeutic option. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier (BBB). To reduce these unwanted effects, we modified the already described BUM690 by exchanging the allyl-into a trifluoro-ethyl group to yield BUM532. Furthermore, we exchanged the nitrogen for oxygen in the trifluoro-ethyl group to yield BUM97. In the intrahippocampal kainic acid mouse model of TLE BUM532 ± phenobarbital (PB), bumetanide ± PB and PB alone significantly reduced hippocampal paroxysmal discharges (HPDs) but not spike trains. By contrast, treatment with BUM97 suppressed HPDs as well as spike trains dose-dependently, more pronounced compared to the other tested compounds and exerted a synergistic anticonvulsant effect with PB. Moreover, at higher doses BUM97 achieved long-lasting reduction of spike trains. In pentylenetetrazole-induced acute seizures only BUM532 combined with a sub-effective dose of PB increased the seizure threshold. No diuretic effects were observed at any dose of the three derivatives. Our data demonstrate the successful optimization of the pharmacological profile of bumetanide and the potential of the improved derivative BUM97 for the treatment of therapy-resistant TLE, in particular in combinatorial drug regimens with a GABA mimetic.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107754DOI Listing
January 2020

Role for Chromatin Remodeling Factor Chd1 in Learning and Memory.

Front Mol Neurosci 2019 23;12. Epub 2019 Jan 23.

Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Precise temporal and spatial regulation of gene expression in the brain is a prerequisite for cognitive processes such as learning and memory. Epigenetic mechanisms that modulate the chromatin structure have emerged as important regulators in this context. While posttranslational modification of histones or the modification of DNA bases have been examined in detail in many studies, the role of ATP-dependent chromatin remodeling factors (ChRFs) in learning- and memory-associated gene regulation has largely remained obscure. Here we present data that implicate the highly conserved chromatin assembly and remodeling factor Chd1 in memory formation and the control of immediate early gene (IEG) response in the hippocampus. We used various paradigms to assess short-and long-term memory in mice bearing a mutated gene that gives rise to an N-terminally truncated protein. Our data demonstrate that the mutation negatively affects long-term object recognition and short- and long-term spatial memory. We found that Chd1 regulates hippocampal expression of the IEG () and () but not and (), because the -mutation led to dysregulation of and expression in naive mice and in mice analyzed at different stages of object location memory (OLM) testing. Of note, Chd1 likely regulates in a direct manner, because chromatin immunoprecipitation (ChIP) assays revealed enrichment of Chd1 upon stimulation at the genomic locus in the hippocampus and in cultured cells. Together these data support a role for Chd1 as a critical regulator of molecular mechanisms governing memory-related processes, and they show that this function involves the N-terminal serine-rich region of the protein.
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http://dx.doi.org/10.3389/fnmol.2019.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351481PMC
January 2019

New Features on the Expression and Trafficking of mGluR1 Splice Variants Exposed by Two Novel Mutant Mouse Lines.

Front Mol Neurosci 2018 3;11:439. Epub 2018 Dec 3.

Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1β are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1β has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering.
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http://dx.doi.org/10.3389/fnmol.2018.00439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287019PMC
December 2018

Proenkephalin Derived Peptides Are Involved in the Modulation of Mitochondrial Respiratory Control During Epileptogenesis.

Front Mol Neurosci 2018 25;11:351. Epub 2018 Sep 25.

Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Epilepsies are a group of common neurological diseases exerting a strong burden on patients and society, often lacking clear etiology and effective therapeutical strategies. Early intervention during the development of epilepsy (epileptogenesis) is of great medical interest, though hampered by poorly characterized epileptogenetic processes. Using the intrahippocampal kainic acid mouse model of temporal lobe epilepsy, we investigated the functional role of the endogenous opioid enkephalin during epileptogenesis. We addressed three sequential questions: (1) How does enkephalin affect seizure threshold and how is it regulated during epileptogenesis? (2) Does enkephalin influence detrimental effects during epileptogenesis? (3) How is enkephalin linked to mitochondrial function during epileptogenesis?. In contrast to other neuropeptides, the expression of enkephalin is not regulated in a seizure dependent manner. The pattern of regulation, and enkephalin's proconvulsive effects suggested it as a potential driving force in epileptogenesis. Surprisingly, enkephalin deficiency aggravated progressive granule cell dispersion in kainic acid induced epileptogenesis. Based on reported beneficial effects of enkephalin on mitochondrial function in hypoxic/ischemic states, we hypothesized that enkephalin may be involved in the adaptation of mitochondrial respiration during epileptogenesis. Using high-resolution respirometry, we observed dynamic improvement of hippocampal mitochondrial respiration after kainic acid-injections in wild-type, but not in enkephalin-deficient mice. Thus, wild-type mice displayed higher efficiency in the use of mitochondrial capacity as compared to enkephalin-deficient mice. Our data demonstrate a Janus-headed role of enkephalin in epileptogenesis. In naive mice, enkephalin facilitates seizures, but in subsequent stages it contributes to neuronal survival through improved mitochondrial respiration.
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http://dx.doi.org/10.3389/fnmol.2018.00351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167428PMC
September 2018

Design, Synthesis, and Pharmacological Evaluation of Novel β2/3 Subunit-Selective γ-Aminobutyric Acid Type A (GABA) Receptor Modulators.

J Med Chem 2019 01 23;62(1):317-341. Epub 2018 Oct 23.

Department of Pharmacology and Toxicology , University of Vienna , Althanstraße 14 , 1090 Vienna , Austria.

Subunit-selective modulation of γ-aminobutyric acid type A receptors (GABAR) is considered to exert fewer side effects compared to unselective clinically used drugs. Here, the β2/3 subunit-selective GABAR modulators valerenic acid (VA) and loreclezole (LOR) guided the synthesis of novel subunit-selective ligands with simplified structures. We studied their effects on GABARs expressed in Xenopus laevis oocytes using two-microelectrode voltage clamp technique. Five compounds showed significantly more efficacious modulation of GABA-evoked currents than VA and LOR with retained potency and selectivity. Compound 18 [( E)-2-Cyano-3-(2,4-dichlorophenyl)but-2-enamide] induced the highest maximal modulation of GABA-induced chloride currents ( E: 3114 ± 242%), while 12 [( Z)-3-(2,4-dichlorophenyl)but-2-enenitrile] displayed the highest potency (EC: 13 ± 2 μM). Furthermore, in hippocampal neurons 12 facilitated phasic and tonic GABAergic inhibition, and in vivo studies revealed significantly more potent protection against pentylenetetrazole (PTZ)-induced seizures compared to VA and LOR. Collectively, compound 12 constitutes a novel, simplified, and subunit-selective GABAR modulator with low-dose anticonvulsant activity.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00859DOI Listing
January 2019

In vivo brain GPCR signaling elucidated by phosphoproteomics.

Science 2018 06;360(6395)

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
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http://dx.doi.org/10.1126/science.aao4927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527112PMC
June 2018

Protein kinase N1 critically regulates cerebellar development and long-term function.

J Clin Invest 2018 05 16;128(5):2076-2088. Epub 2018 Apr 16.

Biocenter, Division of Neurobiochemistry, and.

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber-forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1-/- animals showed a defective PF-Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1-/- Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1-/- Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1-/- Cgcs. In line with our in vitro data, Pkn1-/- mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.
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http://dx.doi.org/10.1172/JCI96165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919825PMC
May 2018

The Opioid System in Temporal Lobe Epilepsy: Functional Role and Therapeutic Potential.

Front Mol Neurosci 2017 7;10:245. Epub 2017 Aug 7.

Department of Pharmacology, Medical University of InnsbruckInnsbruck, Austria.

Temporal lobe epilepsy is considered to be one of the most common and severe forms of focal epilepsies. Patients often develop cognitive deficits and emotional blunting along the progression of the disease. The high incidence of resistance to antiepileptic drugs and a frequent lack of admissibility to surgery poses an unmet medical challenge. In the urgent quest of novel treatment strategies, neuropeptides are interesting candidates, however, their therapeutic potential has not yet been exploited. This review focuses on the functional role of the endogenous opioid system with respect to temporal lobe epilepsy, specifically in the hippocampus. The role of dynorphins and kappa opioid receptors (KOPr) as modulators of neuronal excitability is well understood: both the reduced release of glutamate as well of postsynaptic hyperpolarization were shown in glutamatergic neurons. In line with this, low levels of dynorphin in humans and mice increase the risk of epilepsy development. The role of enkephalins is not understood so well. On one hand, some agonists of the delta opioid receptors (DOPr) display pro-convulsant properties probably through inhibition of GABAergic interneurons. On the other hand, enkephalins play a neuro-protective role under hypoxic or anoxic conditions, most probably through positive effects on mitochondrial function. Despite the supposed absence of endorphins in the hippocampus, exogenous activation of the mu opioid receptors (MOPr) induces pro-convulsant effects. Recently-expanded knowledge of the complex ways opioid receptors ligands elicit their effects (including biased agonism, mixed binding, and opioid receptor heteromers), opens up exciting new therapeutic potentials with regards to seizures and epilepsy. Potential adverse side effects of KOPr agonists may be minimized through functional selectivity. Preclinical data suggest a high potential of such compounds to control seizures, with a strong predictive validity toward human patients. The discovery of DOPr-agonists without proconvulsant potential stimulates the research on the therapeutic use of neuroprotective potential of the enkephalin/DOPr system.
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http://dx.doi.org/10.3389/fnmol.2017.00245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5545604PMC
August 2017

Ablation of Sphingosine 1-Phosphate Receptor Subtype 3 Impairs Hippocampal Neuron Excitability and Spatial Working Memory .

Front Cell Neurosci 2016 7;10:258. Epub 2016 Nov 7.

Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck Innsbruck, Austria.

Understanding the role of the bioactive lipid mediator sphingosine 1-phosphate (S1P) within the central nervous system has recently gained more and more attention, as it has been connected to major diseases such as multiple sclerosis and Alzheimer's disease. Even though much data about the functions of the five S1P receptors has been collected for other organ systems, we still lack a complete understanding for their specific roles, in particular within the brain. Therefore, it was the aim of this study to further elucidate the role of S1P receptor subtype 3 (S1P) and with a special focus on the hippocampus. Using an S1P knock-out mouse model we applied a range of behavioral tests, performed expression studies, and whole cell patch clamp recordings in acute hippocampal slices. We were able to show that S1P deficient mice display a significant spatial working memory deficit within the T-maze test, but not in anxiety related tests. Furthermore, mRNA was expressed throughout the hippocampal formation. Principal neurons in area CA3 lacking S1P showed significantly increased interspike intervals and a significantly decreased input resistance. Upon stimulation with S1P CA3 principal neurons from both wildtype and [Formula: see text] mice displayed significantly increased evoked EPSC amplitudes and decay times, whereas rise times remained unchanged. These results suggest a specific involvement of S1P for the establishment of spatial working memory and neuronal excitability within the hippocampus.
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http://dx.doi.org/10.3389/fncel.2016.00258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097928PMC
November 2016

Restricting calcium currents is required for correct fiber type specification in skeletal muscle.

Development 2016 05 10;143(9):1547-59. Epub 2016 Mar 10.

Department of Physiology and Medical Physics, Medical University Innsbruck, Innsbruck 6020, Austria

Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.
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http://dx.doi.org/10.1242/dev.129676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909858PMC
May 2016

The G-protein biased partial κ opioid receptor agonist 6'-GNTI blocks hippocampal paroxysmal discharges without inducing aversion.

Br J Pharmacol 2016 06 21;173(11):1756-67. Epub 2016 Apr 21.

Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria.

Background And Purpose: With a prevalence of 1-2%, epilepsies belong to the most frequent neurological diseases worldwide. Although antiepileptic drugs are available since several decades, the incidence of patients that are refractory to medication is still over 30%. Antiepileptic effects of κ opioid receptor (κ receptor) agonists have been proposed since the 1980s. However, their clinical use was hampered by dysphoric side effects. Recently, G-protein biased κ receptor agonists were developed, suggesting reduced aversive effects.

Experimental Approach: We investigated the effects of the κ receptor agonist U-50488H and the G-protein biased partial κ receptor agonist 6'-GNTI in models of acute seizures and drug-resistant temporal lobe epilepsy and in the conditioned place avoidance (CPA) test. Moreover, we performed slice electrophysiology to understand the functional mechanisms of 6'-GNTI.

Key Results: As previously shown for U-50488H, 6'-GNTI markedly increased the threshold for pentylenetetrazole-induced seizures. All treated mice displayed reduced paroxysmal activity in response to U-50488H (20 mg·kg(-1) ) or 6'-GNTI (10-30 nmoles) treatment in the mouse model of intra-hippocampal injection of kainic acid. Single cell recordings on hippocampal pyramidal cells revealed enhanced inhibitory signalling as potential mechanisms causing the reduction of paroxysmal activity. Effects of 6'-GNTI were blocked in both seizure models by the κ receptor antagonist 5'-GNTI. Moreover, 6'-GNTI did not induce CPA, a measure of aversive effects, while U-50488H did.

Conclusions And Implications: Our data provide the proof of principle that anticonvulsant/antiseizure and aversive effects of κ receptor activation can be pharmacologically separated in vivo.
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http://dx.doi.org/10.1111/bph.13474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867738PMC
June 2016

Metallothioneins and renal ageing.

Nephrol Dial Transplant 2016 09 3;31(9):1444-52. Epub 2016 Feb 3.

Department of Internal Medicine IV (Nephrology and Hypertension), Medical University Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.

Background: Human lifespan is increasing continuously and about one-third of the population >70 years of age suffers from chronic kidney disease. The pathophysiology of the loss of renal function with ageing is unclear.

Methods: We determined age-associated gene expression changes in zero-hour biopsies of deceased donor kidneys without laboratory signs of impaired renal function, defined as a last serum creatinine >0.96 mg/dL in females and >1.18 mg/dL in males, using microarray technology and the Significance Analysis of Microarrays routine. Expression changes of selected genes were confirmed by quantitative polymerase chain reaction and in situ hybridization and immunohistochemistry for localization of respective mRNA and protein. Functional aspects were examined in vitro.

Results: Donors were classified into three age groups (<40, 40-59 and >59 years; Groups 1, 2 and 3, respectively). In Group 3 especially, genes encoding for metallothionein (MT) isoforms were more significantly expressed when compared with Group 1; localization studies revealed predominant staining in renal proximal tubular cells. RPTEC/TERT1 cells overexpressing MT2A were less susceptible towards cadmium chloride-induced cytotoxicity and hypoxia-induced apoptosis, both models for increased generation of reactive oxygen species.

Conclusions: Increased expression of MTs in the kidney with ageing might be a protective mechanism against increased oxidative stress, which is closely related to the ageing process. Our findings indicate that MTs are functionally involved in the pathophysiology of ageing-related processes.
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http://dx.doi.org/10.1093/ndt/gfv451DOI Listing
September 2016

Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

J Comp Neurol 2016 07 15;524(10):2093-116. Epub 2015 Dec 15.

Department of Pharmacology, Medical University of Innsbruck, 6020, Innsbruck, Austria.

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/cne.23934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982087PMC
July 2016

Sprouty2 and -4 hypomorphism promotes neuronal survival and astrocytosis in a mouse model of kainic acid induced neuronal damage.

Hippocampus 2016 May 27;26(5):658-67. Epub 2015 Nov 27.

Division of Neuroanatomy, Department of Anatomy Histology and Embryology, Medical University, Innsbruck, 6020, Innsbruck, Austria.

Sprouty (Spry) proteins play a key role as negative feedback inhibitors of the Ras/Raf/MAPK/ERK pathway downstream of various receptor tyrosine kinases. Among the four Sprouty isoforms, Spry2 and Spry4 are expressed in the hippocampus. In this study, possible effects of Spry2 and Spry4 hypomorphism on neurodegeneration and seizure thresholds in a mouse model of epileptogenesis was analyzed. The Spry2/4 hypomorphs exhibited stronger ERK activation which was limited to the CA3 pyramidal cell layer and to the hilar region. The seizure threshold of Spry2/4(+/-) mice was significantly reduced at naive state but no difference to wildtype mice was observed 1 month following KA treatment. Histomorphological analysis revealed that dentate granule cell dispersion (GCD) was diminished in Spry2/4(+/-) mice in the subchronic phase after KA injection. Neuronal degeneration was reduced in CA1 and CA3 principal neuron layers as well as in scattered neurons of the contralateral CA1 and hilar regions. Moreover, Spry2/4 reduction resulted in enhanced survival of somatostatin and neuropeptide Y expressing interneurons. GFAP staining intensity and number of reactive astrocytes markedly increased in lesioned areas of Spry2/4(+/-) mice as compared with wildtype mice. Taken together, although the seizure threshold is reduced in naive Spry2/4(+/-) mice, neurodegeneration and GCD is mitigated following KA induced hippocampal lesions, identifying Spry proteins as possible pharmacological targets in brain injuries resulting in neurodegeneration. The present data are consistent with the established functions of the ERK pathway in astrocyte proliferation as well as protection from neuronal cell death and suggest a novel role of Spry proteins in the migration of differentiated neurons.
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http://dx.doi.org/10.1002/hipo.22549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949526PMC
May 2016

Differences in mitochondrial function in homogenated samples from healthy and epileptic specific brain tissues revealed by high-resolution respirometry.

Mitochondrion 2015 Nov 26;25:104-12. Epub 2015 Oct 26.

D. Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University Innsbruck, Anichstr. 35, 6020 Innsbruck, Austria; OROBOROS INSTRUMENTS, Schöpfstr. 18, 6020 Innsbruck, Austria. Electronic address:

Mitochondrial dysfunction and oxidative stress are strongly implicated in neurodegenerative diseases and epilepsy. Strikingly, neurodegenerative diseases show regional specificity in vulnerability and follow distinct patterns of neuronal loss. A challenge is to understand, why mitochondria fail in particular brain regions under specific pathological conditions. A potential explanation could be provided by regional or cellular specificity of mitochondrial function. We applied high-resolution respirometry to analyze the integrated Complex I- and II (CI and CII)-linked respiration, the activity of Complex IV, and the combined CI&II-linked oxidative phosphorylation (OXPHOS)- and electron-transfer system (ETS)-capacity in microsamples obtained from distinct regions of the mouse brain. We compared different approaches to assess mitochondrial density and suggest flux control ratios as a valid method to normalize respiration to mitochondrial density. This approach revealed significant differences of CI- and CII-linked OXPHOS capacity and coupling control between motor cortex, striatum, hippocampus and pons of naïve mice. CI-linked respiration was highest in motor cortex, while CII-linked respiration predominated in the striatum. To investigate if this method could also determine differences in normal and disease states within the same brain region, we compared hippocampal homogenates in a chronic epilepsy model. Three weeks after stereotaxic injection of kainate, there was a down-regulation of CI- and upregulation of CII-linked respiration in the resulting epileptic ipsilateral hippocampus compared to the contralateral one. In summary, respirometric OXPHOS analysis provides a very sensitive diagnostic approach using small amounts of distinct brain tissues. In a single assay, information is obtained on numerous OXPHOS parameters as indicators of tissue-specific mitochondrial performance.
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http://dx.doi.org/10.1016/j.mito.2015.10.007DOI Listing
November 2015

Identification of potential novel interaction partners of the sodium-activated potassium channels Slick and Slack in mouse brain.

Biochem Biophys Rep 2015 Dec 9;4:291-298. Epub 2015 Oct 9.

Division of Molecular and Cellular Pharmacology, Medical University of Innsbruck, Peter-Mayr Str 1, Innsbruck, Austria.

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are paralogous channels of the Slo family of high-conductance potassium channels. Slick and Slack channels are widely distributed in the mammalian CNS and they play a role in slow afterhyperpolarization, generation of depolarizing afterpotentials and in setting and stabilizing the resting potential. In the present study we used a combined approach of (co)-immunoprecipitation studies, Western blot analysis, double immunofluorescence and mass spectrometric sequencing in order to investigate protein-protein interactions of the Slick and Slack channels. The data strongly suggest that Slick and Slack channels co-assemble into identical cellular complexes. Double immunofluorescence experiments revealed that Slick and Slack channels co-localize in distinct mouse brain regions. Moreover, we identified the small cytoplasmic protein beta-synuclein and the transmembrane protein 263 (TMEM 263) as novel interaction partners of both, native Slick and Slack channels. In addition, the inactive dipeptidyl-peptidase (DPP 10) and the synapse associated protein 102 (SAP 102) were identified as constituents of the native Slick and Slack channel complexes in the mouse brain. This study presents new insights into protein-protein interactions of native Slick and Slack channels in the mouse brain.
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http://dx.doi.org/10.1016/j.bbrep.2015.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669359PMC
December 2015

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

Mol Cell Neurosci 2015 Jul 20;67:104-15. Epub 2015 Jun 20.

University of Zagreb School of Medicine, Croatian Institute for Brain Research, Zagreb, Croatia. Electronic address:

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.
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http://dx.doi.org/10.1016/j.mcn.2015.06.009DOI Listing
July 2015

Reacquisition of cocaine conditioned place preference and its inhibition by previous social interaction preferentially affect D1-medium spiny neurons in the accumbens corridor.

Front Behav Neurosci 2014 24;8:317. Epub 2014 Sep 24.

Experimental Psychiatry Unit, Innsbruck Medical University Innsbruck, Austria ; Department of Psychology, Leopold-Franzens University of Innsbruck Innsbruck, Austria.

We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), differentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. We also investigated the involvement of the lateral accumbens core and the dorsal caudate putamen. The anterior cingulate 1 (Cg1) region served as a negative control. Contrary to our expectations, we found that all regions of the accumbens corridor showed increased expression of the early growth response protein 1 (EGR1, Zif268) in rats 2 h after reacquisition of CPP for cocaine after a history of cocaine CPP acquisition and extinction. Previous counterconditioning with dyadic social interaction inhibited both the reacquisition of cocaine CPP and the activation of the whole accumbens corridor. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less affected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders.
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http://dx.doi.org/10.3389/fnbeh.2014.00317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174134PMC
October 2014

GPER1 (GPR30) knockout mice display reduced anxiety and altered stress response in a sex and paradigm dependent manner.

Horm Behav 2014 Sep 16;66(4):628-36. Epub 2014 Sep 16.

Department of Pharmacology, Innsbruck Medical University, Peter-Mayr-Str. 1a, A-6020 Innsbruck, Austria. Electronic address:

The putative estrogen receptor GPER1 (the former orphan receptor GPR30) is discussed to be involved in emotional and cognitive functions and stress control. We recently described the induction of anxiety-like effects by the GPER1 agonist G-1 upon systemic injection into mice. To contribute to a better understanding of the role of GPER1 in anxiety and stress, we investigated germ-line GPER1 deficient mice. Our experiments revealed marked differences between the sexes. A mild but consistent phenotype of increased exploratory drive was observed in the home cage, the elevated plus maze and the light-dark choice test in male GPER1 KO mice. In contrast, female GPER1-KO mice displayed a less pronounced phenotype in these tests. Estrous-stage dependent mild anxiolytic-like effects were observed solely in the open field test. Notably, we observed a strong shift in acute stress coping behavior in the tail suspension test and basal corticosterone levels in different phases of the estrous cycle in female GPER1-KO mice. Our data, in line with previous reports, suggest that GPER1 is involved in anxiety and stress control. Surprisingly, its effects appear to be stronger in male than female mice.
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http://dx.doi.org/10.1016/j.yhbeh.2014.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213071PMC
September 2014

A cycloartane glycoside derived from Actaea racemosa L. modulates GABAA receptors and induces pronounced sedation in mice.

J Pharmacol Exp Ther 2014 Nov 26;351(2):234-42. Epub 2014 Aug 26.

Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria (B.S., S.K., S.H.); Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria (I.K., C.S.); and Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria (S.S.C., H.S.).

23-O-Acetylshengmanol 3-O-β-D-xylopyranoside (Ac-SM) isolated from Actaea racemosa L.-an herbal remedy for the treatment of mild menopausal disorders-has been recently identified as a novel efficacious modulator of GABAA receptors composed of α1-, β2-, and γ2S-subunits. In the present study, we analyzed a potential subunit-selective modulation of GABA-induced chloride currents (IGABA) at GABA concentrations eliciting 3-8% of the maximal GABA response (EC3-8) through nine GABAA receptor isoforms expressed in Xenopus laevis oocytes by Ac-SM with two-microelectrode voltage clamp and behavioral effects 30 minutes after intraperitoneal application in a mouse model. Efficacy of IGABA enhancement by Ac-SM displayed a mild α-subunit dependence with α2β2γ2S (maximal IGABA potentiation [Emax] = 1454 ± 97%) and α5β2γ2S (Emax = 1408 ± 87%) receptors being most efficaciously modulated, followed by slightly weaker IGABA enhancement through α1β2γ2S (Emax = 1187 ± 166%), α3β2γ2S (Emax = 1174 ± 218%), and α6β2γ2S (Emax = 1171 ± 274%) receptors and less pronounced effects on receptors composed of α4β2γ2S (Emax = 752 ± 53%) subunits, whereas potency was not affected by the subunit composition (EC50 values ranging from α1β2γ2S = 35.4 ± 12.3 µM to α5β2γ2S = 50.9 ± 11.8 µM). Replacing β2- with β1- or β3-subunits as well as omitting the γ2S-subunit affected neither efficacy nor potency of IGABA enhancement by Ac-SM. Ac-SM shifted the GABA concentration-response curve toward higher GABA sensitivity (about 3-fold) and significantly increased the maximal GABA response by 44 ± 13%, indicating a pharmacological profile distinct from a pure allosteric GABAA receptor modulator. In mice, Ac-SM significantly reduced anxiety-related behavior in the elevated plus maze test at a dose of 0.6 mg/kg, total ambulation in the open field test at doses ≥6 mg/kg, stress-induced hyperthermia at doses ≥0.6 mg/kg, and significantly elevated seizure threshold at doses ≥20 mg/kg body weight. High efficacy and long biologic half-life of Ac-SM suggest that potential cumulative sedative side effects upon repetitive intake of A. racemosa L. preparations might not be negligible.
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http://dx.doi.org/10.1124/jpet.114.218024DOI Listing
November 2014
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