S1-A FEPS European Young Physiologists Symposium
S1-A1 Cell type-specific subcellular distribution of ion channels in the central nervous system A. Lőrincz Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
Neuronal excitability is underlined by active conductances generated by voltage-gated ion channels. The functional impact of a voltage-gated ion channel is highly affected by its precise location in the axo-somato-dendritic domains of nerve cells. In this presentation, I will demonstrate that the organizing rules governing the subcellular distribution of voltage-gated Na+ (Nav) and K+ (Kv) channels are more complicated than previously believed. In the axon initial segment (AIS) of neurons, high density of Nav channels ensure low threshold for action potential initiation and Kv channels are key regulators of action potential repolarization, firing rate and pattern. Highly sensitive immunohistochemical methods allowed us to reveal heterogeneity in the density and distribution of distinct Nav and Kv channel subunits within the AIS of different types of nerve cells. This precise arrangement is likely to contribute to the diversity of firing properties observed among central neurons. In dendrites, active invasion of action potentials generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In pyramidal cells this action potential backpropagation is supported by dendritic Nav channels. Employing the high resolution SDS-digested freezefracture replica-labelling method allowed us to reveal quantitative differences in the subcellular distribution of the predominant Nav channels in the hippocampal CA1 pyramidal cells. Our results suggest that variability in the subcellular distribution of ion channels is a way of increasing neuronal diversity in the central nervous system.
S1-A2 Rapid generation of human neurons modeling neuropsychiatric disorders
for
T. Danko Department of Molecular and Cellular Physiology, Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, Stanford, USA
1
Previous studies demonstrated the availability of various methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into induced neurons (iN). Alternatively, human fibroblasts can also be directly converted into induced neuronal (iN) cells. These studies also described that with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. However, with the advent of patient- derived, disease-specific iPSC technology, there is an enormous potential in iN cells for studying the pathogenesis of various, poorly understood neurological disorders, such as autism, schizophrenia or Alzheimer’s disease. Furthermore, this technology also enables researchers to design drug screening systems, and to produce neurons for purposes of regenerative medicine. This requires the capability of large-scale production of human iN cells and with a high yield, and also necessitates the generation of iN cells that readily form synapses. Moreover, such goals could be facilitated by a high degree of reproducibility independent of the starting cell line and by production of a relatively homogeneous population of functional iN cells for experimental purposes. We performed lentivirus-mediated exogenous over-expression of the transcription factor neurogenin 2 (Ngn2) in human ESCs and iPSCs to derive induced neurons. Potential differences in neuronal maturation, morphology, synapse formation and synaptic function was then quantified by various techniques including electrophysiology. We show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. Our approach enables large-scale studies of human neurons for goals such as analyses of human diseases, examination of human-specific genes, and drug screening.
S1-A3 Descending effect on spinal nociception by amygdaloid glutamate varies with the submodality of noxious test stimulation N. Bourbia, B. Sagalajev, A. Pertovaara Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland
Amygdala has an important role in the processing of primary emotions, such as fear. Additionally, amygdala is involved in processing and modulation of pain. While the amygdala, particularly its central nucleus (CeA), has been shown to contribute to pain control, the descending pain regulation by the CeA is still only partly characterized. Here heat and mechanical nociception was tested in both hind limbs of healthy rats with a chronic guide cannula for microinjection of glutamate into the CeA of the left or right hemisphere. The aim was to assess whether the descending pain regulatory effect by glutamate in the amygdala varies with the
submodality or the body side of nociceptive testing, brain hemisphere or the amygdaloid glutamate receptor. Motor performance was assessed with the Rotarod test. Amygdaloid glutamate, independent of the treated hemisphere, produced a dose-related heat and mechanical antinociception that varied with the submodality of testing. Heat antinociception was short lasting (minutes), bilateral and not reversed by blocking the amygdaloid NMDA receptor with MK-801. In contrast, mechanical antinociception lasted longer (>20 min), was predominantly contralateral and reversed by blocking the amygdaloid NMDA receptor. At an antinociceptive dose, amygdaloid glutamate failed to influence motor performance. The results indicate that independent of the brain hemisphere, the spatial extent and duration of the descending antinociceptive effect induced by amygdaloid glutamate varies with the amygdaloid glutamate receptor and the submodality of pain.
and different inhibitors: in LV of L-NAME group and BS of 7-NI group. We can conclude that radical signaling and antioxidant response was mainly influenced in L-NAME group and this response was more pronounced in LV of young rats and in BS of adult WR rats. Supported by SAS-NSC JRP 2010/1, APVV-0348-12, Ministry of Health - 2012/51-SAV-1, SR.
S1-A5 Quantitative pilomotor axon-reflex test to assess autonomic dysfunction in Parkinson's disease T. Siepmann1,2, E. Frenz2, W. Kirch1, B. Min-Woo Illigens1,3 1
Institute of Clinical Pharmacology, Carl Gustav Carus University Hospital, Dresden University of Technology, Dresden, Germany; Department of Neurology, Carl Gustav Carus University Hospital, Dresden University of Technology, Dresden, Germany; 3 Department of Neurology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, Massachusetts, U.S.A. 2
S1-A4 The effect of NOS inhibitors on radical signaling and antioxidant response in Wistar rats M. Majzúnová1, Z. Pakanová2, P. Bališ1, M. Drobná1, I. Dovinová1 1
Institute of Normal and Pathologigal Physiology Slovak Academy of Sciences, Bratislava, SR, 2I nstitute of Chemistry Slovak Academy of Sciences, Bratislava, SR
Despite of many experimental studies, the effect of NOS inhibition on activation of radical pathway (AT1R-NADPH oxidase-superoxide) is not yet sufficiently clarified. In our study, we used two inhibitors of NO-synthase, specific inhibitor of nNOS: 7-nitroindazole (7-NI) and nonspecific inhibitor of NOS: NG-nitro-L-arginine-methyl ester (L-NAME). The aim of our study was to determine changes in free radical signaling, antioxidant and detoxification response in left ventricular of heart (LV) and brainstem (BS) of young and adult Wistar rats (WR) during NO-deficiency. Young (4 weeks) and adult (10 weeks) WR were treated with 7-NI (10 mg/kg/day), L-NAME (50 mg/kg/day) or drinking water (Control) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Level of superoxide was determined by Lucigenin-enhanced chemiluminescence. Expression of genes (AT1R, p22phox subunit of NADPH oxidase, SOD and NOS isoforms, HO-1, MDR1a and housekeeper GAPDH) were identified by realtime PCR. Activity of NOS was detected by conversion of [3H]-L-arginine on [3H]-L-citrulline and activity of SOD was measured by UV VIS spectroscopy. Blood pressure was increased after L-NAME treatment in young and adult WR. Activity of NOS was decreased only after L-NAME application (young and adult rats) without changes in eNOS and nNOS mRNA. Both inhibitors decreased superoxide level in LV of young animals without changes in AT1R or p22phox mRNA expression, while in adult rats only L-NAME affected AT1R and p22phox mRNA elevation in BS. Increased SOD activity positively correlated with SOD3 and HO-1 mRNA elevation only in this group, where we found also MDR1a mRNA elevation. Increase in MDR1a was observed also in young rats in different tissues
BACKGROUND: Early detection of Parkinson’s disease (PD)
contributes to treatment success but is limited by the lack of techniques to assess early PD symptoms such as autonomic neuropathy. We recently demonstrated that peripheral autonomic nerve function can be assessed through iontophoresis of phenylephrine to elicit axon- reflex mediated piloerection in an indirect skin region outside the region of iontophoretic stimulation using the quantitative pilomotor axon-reflex test (QPART). (1) AIM. This study aimed to assess the hypothesis that QPART is a valid measure of autonomic pilomotor nerve dysfunction in early PD. METHODS: Six healthy subjects and six age matched PD patients (Hoehn and Yahr stages I-II) participated in a controlled study. Piloerection was stimulated by iontophoresis of 1% phenylephrine on the dorsal forearm. Silicone impressions of resulting goose bumps were digitally quantified by number and area. Comparative automomic measures included heart rate variability (HRV)-analysis, cutaneous vasomotor function assessment using laser Doppler flowmetry, and cutaneous sudomotor function measurement using skin conductance resoponses. RESULTS: In PD patients, the number of impressions in the indirect region was lower compared with healthy subjects (19.0±7.9 PD vs. 27.3±15.5 subjects, mean±SD, p ˂0.05). Sudomotor function was attenuated in PD patients compared with healthy controls (p ˂0.01) whereas we observed nonsignificant trends toward decreased HRV and vasomotor function in PD patients (p=n.s.). CONCLUSIONS: This study demonstrates that axon-reflex pilomotor responses are impaired in early PD stages compared with healthy controls, consistent with impaired sudomotor function, suggesting that QPART is a valid test to detect early autonomic nerve dysfunction in PD. (1) Siepmann T, Gibbons CH, Illigens BM, et al. Quantitative pilomotor axon reflex test: a novel test of pilomotor function. Arch Neurol. 2012;69(11):1488-92.
2
S1-A6 Reprograming of liver alternative p38-mediated neutrophil migration
metabolism modulation
by of
G. Sabio Assistant Professor: Stress kinases in Diabetes, Cancer and Cardiovascular Disease Department Vascular Biology and Inflammation Centro Nacional de Investigaciones Cardiovasculares Carlos III C/ Melchor Fernández Almagro, 3 28029 Madrid (Spain)
Nonalcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ), have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to dietinduced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential therapeutic target for NAFLD treatment.
The voltage dependence of the normalized fluorescence of the calcium transients in response to 100 ms long membrane depolarizations ranging between -60 and +30 mV, with 10 mV increments were not statistically different and were well fitted with a Boltzmann distribution (V0.5: -23.22±1.35 mV vs 24.15±0.77 mV with respective k values of 6.14±1.15 vs 6.93±0.65). To both elicit the release response and substantially deplete the SR of calcium and induce muscle fatigue, the evolution of [Ca2+]i during a train of eight longlasting depolarizations (200 ms) to a maximally activating voltage (+30 mV) were monitored. The release flux parameters and the amount of Ca2+ released during these pulses were decreased in the mutant muscles. Though myostatin deficiency is an unwanted condition by itself, understanding the underlying mechanisms may help developing new safe strategies to cure muscle wasting diseases and alleviate symptoms of muscle weakness. Funded by: OTKA PD-108476
S1-A8 MiRNA-24 antagonism prevents renal ischemia reperfusion injury J. M. Lorenzen1,2, T. Kaucsár1,3, C.Schauerte1, R. Schmitt2, S. Rong2, A. Hübner1, K. Scherf1, J. Fiedler1, F. Martino1, K. Regalla1, M. Kölling1, I. Sörensen2, H. Hinz4, J. Heineke4, E. v Rooij5, H. Haller2, T. Thum1,6 1
Institute of Molecular and Translational Therapeutic Strategies, Hanover Medical School, Hanover, Germany Department of Nephrology, Hanover Medical School, Hanover, Germany 3 Institute of Pathophysiology, Semmelweis University, Budapest, Hungary 4 Department of Cardiology and Angiology, Hanover Medical School, Hanover, Germany 5 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Utrecht, The Netherlands 6 National Heart and Lung Institute, Imperial College London, UK 2
S1-A7 A hypermuscular mouse model to study SOCE and muscle fatigue M. Sztretye University of Debrecen, Department of Physiology, Debrecen, Hungary
In our mouse model, a naturally occuring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (MstnCmpt-dl1Abc) labeled by a tremendous increase in body weight along with signs of muscle weakness and easier fatiguability. To characterize and better understand the mechanism underlying these alterations, we monitored store-operated Ca2+-entry (SOCE) and also measured the cytosolic [Ca2+] (rhod2) using whole cell voltage clamp technique. Since STIM1 and Orai1 endogenous protein levels in the mutant flexor digitorum brevis (FDB) muscles were reduced by ~30% we hypothesized that SOCE, a potential candidate to assist in the longer–term maintenance of EC coupling may be consequently altered. Enzymatically isolated fluo-8 AM loaded FDB fibers were used. To elicit a massive SR Ca2+release a RyR1 agonist (4-chloro-meta-cresol, 4-CmC) was applied in a Ca2+ free medium and in the presence of the SR Ca2+ pump inhibitor (thapsigargin, TG). The above cocktail triggered a deep transient store depletion which in turn switched on SOCE. The peak of this slow Ca2+ transient was normalized to the peak of the SR Ca2+ release transient (0.63±0.09, n=18 vs 0.32±0.08*, n=11, p≤0.02).
3
Ischemia-reperfusion (I/R) injury of the kidney is one of the major causes of acute kidney injury. MicroRNAs are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R-injury. MiR-24 is upregulated in the kidney following I/R-injury of mice (4.7 fold, p
S1-A1 Cell type-specific subcellular distribution of ion channels in the central nervous system A. Lőrincz Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
Neuronal excitability is underlined by active conductances generated by voltage-gated ion channels. The functional impact of a voltage-gated ion channel is highly affected by its precise location in the axo-somato-dendritic domains of nerve cells. In this presentation, I will demonstrate that the organizing rules governing the subcellular distribution of voltage-gated Na+ (Nav) and K+ (Kv) channels are more complicated than previously believed. In the axon initial segment (AIS) of neurons, high density of Nav channels ensure low threshold for action potential initiation and Kv channels are key regulators of action potential repolarization, firing rate and pattern. Highly sensitive immunohistochemical methods allowed us to reveal heterogeneity in the density and distribution of distinct Nav and Kv channel subunits within the AIS of different types of nerve cells. This precise arrangement is likely to contribute to the diversity of firing properties observed among central neurons. In dendrites, active invasion of action potentials generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In pyramidal cells this action potential backpropagation is supported by dendritic Nav channels. Employing the high resolution SDS-digested freezefracture replica-labelling method allowed us to reveal quantitative differences in the subcellular distribution of the predominant Nav channels in the hippocampal CA1 pyramidal cells. Our results suggest that variability in the subcellular distribution of ion channels is a way of increasing neuronal diversity in the central nervous system.
S1-A2 Rapid generation of human neurons modeling neuropsychiatric disorders
for
T. Danko Department of Molecular and Cellular Physiology, Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, Stanford, USA
1
Previous studies demonstrated the availability of various methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into induced neurons (iN). Alternatively, human fibroblasts can also be directly converted into induced neuronal (iN) cells. These studies also described that with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. However, with the advent of patient- derived, disease-specific iPSC technology, there is an enormous potential in iN cells for studying the pathogenesis of various, poorly understood neurological disorders, such as autism, schizophrenia or Alzheimer’s disease. Furthermore, this technology also enables researchers to design drug screening systems, and to produce neurons for purposes of regenerative medicine. This requires the capability of large-scale production of human iN cells and with a high yield, and also necessitates the generation of iN cells that readily form synapses. Moreover, such goals could be facilitated by a high degree of reproducibility independent of the starting cell line and by production of a relatively homogeneous population of functional iN cells for experimental purposes. We performed lentivirus-mediated exogenous over-expression of the transcription factor neurogenin 2 (Ngn2) in human ESCs and iPSCs to derive induced neurons. Potential differences in neuronal maturation, morphology, synapse formation and synaptic function was then quantified by various techniques including electrophysiology. We show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. Our approach enables large-scale studies of human neurons for goals such as analyses of human diseases, examination of human-specific genes, and drug screening.
S1-A3 Descending effect on spinal nociception by amygdaloid glutamate varies with the submodality of noxious test stimulation N. Bourbia, B. Sagalajev, A. Pertovaara Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland
Amygdala has an important role in the processing of primary emotions, such as fear. Additionally, amygdala is involved in processing and modulation of pain. While the amygdala, particularly its central nucleus (CeA), has been shown to contribute to pain control, the descending pain regulation by the CeA is still only partly characterized. Here heat and mechanical nociception was tested in both hind limbs of healthy rats with a chronic guide cannula for microinjection of glutamate into the CeA of the left or right hemisphere. The aim was to assess whether the descending pain regulatory effect by glutamate in the amygdala varies with the
submodality or the body side of nociceptive testing, brain hemisphere or the amygdaloid glutamate receptor. Motor performance was assessed with the Rotarod test. Amygdaloid glutamate, independent of the treated hemisphere, produced a dose-related heat and mechanical antinociception that varied with the submodality of testing. Heat antinociception was short lasting (minutes), bilateral and not reversed by blocking the amygdaloid NMDA receptor with MK-801. In contrast, mechanical antinociception lasted longer (>20 min), was predominantly contralateral and reversed by blocking the amygdaloid NMDA receptor. At an antinociceptive dose, amygdaloid glutamate failed to influence motor performance. The results indicate that independent of the brain hemisphere, the spatial extent and duration of the descending antinociceptive effect induced by amygdaloid glutamate varies with the amygdaloid glutamate receptor and the submodality of pain.
and different inhibitors: in LV of L-NAME group and BS of 7-NI group. We can conclude that radical signaling and antioxidant response was mainly influenced in L-NAME group and this response was more pronounced in LV of young rats and in BS of adult WR rats. Supported by SAS-NSC JRP 2010/1, APVV-0348-12, Ministry of Health - 2012/51-SAV-1, SR.
S1-A5 Quantitative pilomotor axon-reflex test to assess autonomic dysfunction in Parkinson's disease T. Siepmann1,2, E. Frenz2, W. Kirch1, B. Min-Woo Illigens1,3 1
Institute of Clinical Pharmacology, Carl Gustav Carus University Hospital, Dresden University of Technology, Dresden, Germany; Department of Neurology, Carl Gustav Carus University Hospital, Dresden University of Technology, Dresden, Germany; 3 Department of Neurology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, Massachusetts, U.S.A. 2
S1-A4 The effect of NOS inhibitors on radical signaling and antioxidant response in Wistar rats M. Majzúnová1, Z. Pakanová2, P. Bališ1, M. Drobná1, I. Dovinová1 1
Institute of Normal and Pathologigal Physiology Slovak Academy of Sciences, Bratislava, SR, 2I nstitute of Chemistry Slovak Academy of Sciences, Bratislava, SR
Despite of many experimental studies, the effect of NOS inhibition on activation of radical pathway (AT1R-NADPH oxidase-superoxide) is not yet sufficiently clarified. In our study, we used two inhibitors of NO-synthase, specific inhibitor of nNOS: 7-nitroindazole (7-NI) and nonspecific inhibitor of NOS: NG-nitro-L-arginine-methyl ester (L-NAME). The aim of our study was to determine changes in free radical signaling, antioxidant and detoxification response in left ventricular of heart (LV) and brainstem (BS) of young and adult Wistar rats (WR) during NO-deficiency. Young (4 weeks) and adult (10 weeks) WR were treated with 7-NI (10 mg/kg/day), L-NAME (50 mg/kg/day) or drinking water (Control) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Level of superoxide was determined by Lucigenin-enhanced chemiluminescence. Expression of genes (AT1R, p22phox subunit of NADPH oxidase, SOD and NOS isoforms, HO-1, MDR1a and housekeeper GAPDH) were identified by realtime PCR. Activity of NOS was detected by conversion of [3H]-L-arginine on [3H]-L-citrulline and activity of SOD was measured by UV VIS spectroscopy. Blood pressure was increased after L-NAME treatment in young and adult WR. Activity of NOS was decreased only after L-NAME application (young and adult rats) without changes in eNOS and nNOS mRNA. Both inhibitors decreased superoxide level in LV of young animals without changes in AT1R or p22phox mRNA expression, while in adult rats only L-NAME affected AT1R and p22phox mRNA elevation in BS. Increased SOD activity positively correlated with SOD3 and HO-1 mRNA elevation only in this group, where we found also MDR1a mRNA elevation. Increase in MDR1a was observed also in young rats in different tissues
BACKGROUND: Early detection of Parkinson’s disease (PD)
contributes to treatment success but is limited by the lack of techniques to assess early PD symptoms such as autonomic neuropathy. We recently demonstrated that peripheral autonomic nerve function can be assessed through iontophoresis of phenylephrine to elicit axon- reflex mediated piloerection in an indirect skin region outside the region of iontophoretic stimulation using the quantitative pilomotor axon-reflex test (QPART). (1) AIM. This study aimed to assess the hypothesis that QPART is a valid measure of autonomic pilomotor nerve dysfunction in early PD. METHODS: Six healthy subjects and six age matched PD patients (Hoehn and Yahr stages I-II) participated in a controlled study. Piloerection was stimulated by iontophoresis of 1% phenylephrine on the dorsal forearm. Silicone impressions of resulting goose bumps were digitally quantified by number and area. Comparative automomic measures included heart rate variability (HRV)-analysis, cutaneous vasomotor function assessment using laser Doppler flowmetry, and cutaneous sudomotor function measurement using skin conductance resoponses. RESULTS: In PD patients, the number of impressions in the indirect region was lower compared with healthy subjects (19.0±7.9 PD vs. 27.3±15.5 subjects, mean±SD, p ˂0.05). Sudomotor function was attenuated in PD patients compared with healthy controls (p ˂0.01) whereas we observed nonsignificant trends toward decreased HRV and vasomotor function in PD patients (p=n.s.). CONCLUSIONS: This study demonstrates that axon-reflex pilomotor responses are impaired in early PD stages compared with healthy controls, consistent with impaired sudomotor function, suggesting that QPART is a valid test to detect early autonomic nerve dysfunction in PD. (1) Siepmann T, Gibbons CH, Illigens BM, et al. Quantitative pilomotor axon reflex test: a novel test of pilomotor function. Arch Neurol. 2012;69(11):1488-92.
2
S1-A6 Reprograming of liver alternative p38-mediated neutrophil migration
metabolism modulation
by of
G. Sabio Assistant Professor: Stress kinases in Diabetes, Cancer and Cardiovascular Disease Department Vascular Biology and Inflammation Centro Nacional de Investigaciones Cardiovasculares Carlos III C/ Melchor Fernández Almagro, 3 28029 Madrid (Spain)
Nonalcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38γ and p38δ), have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38δ is elevated in livers of patients with NAFLD and that mice lacking p38γ/δ in myeloid cells are resistant to dietinduced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38γ/δ-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38γ and p38δ in myeloid cells provide a potential therapeutic target for NAFLD treatment.
The voltage dependence of the normalized fluorescence of the calcium transients in response to 100 ms long membrane depolarizations ranging between -60 and +30 mV, with 10 mV increments were not statistically different and were well fitted with a Boltzmann distribution (V0.5: -23.22±1.35 mV vs 24.15±0.77 mV with respective k values of 6.14±1.15 vs 6.93±0.65). To both elicit the release response and substantially deplete the SR of calcium and induce muscle fatigue, the evolution of [Ca2+]i during a train of eight longlasting depolarizations (200 ms) to a maximally activating voltage (+30 mV) were monitored. The release flux parameters and the amount of Ca2+ released during these pulses were decreased in the mutant muscles. Though myostatin deficiency is an unwanted condition by itself, understanding the underlying mechanisms may help developing new safe strategies to cure muscle wasting diseases and alleviate symptoms of muscle weakness. Funded by: OTKA PD-108476
S1-A8 MiRNA-24 antagonism prevents renal ischemia reperfusion injury J. M. Lorenzen1,2, T. Kaucsár1,3, C.Schauerte1, R. Schmitt2, S. Rong2, A. Hübner1, K. Scherf1, J. Fiedler1, F. Martino1, K. Regalla1, M. Kölling1, I. Sörensen2, H. Hinz4, J. Heineke4, E. v Rooij5, H. Haller2, T. Thum1,6 1
Institute of Molecular and Translational Therapeutic Strategies, Hanover Medical School, Hanover, Germany Department of Nephrology, Hanover Medical School, Hanover, Germany 3 Institute of Pathophysiology, Semmelweis University, Budapest, Hungary 4 Department of Cardiology and Angiology, Hanover Medical School, Hanover, Germany 5 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Utrecht, The Netherlands 6 National Heart and Lung Institute, Imperial College London, UK 2
S1-A7 A hypermuscular mouse model to study SOCE and muscle fatigue M. Sztretye University of Debrecen, Department of Physiology, Debrecen, Hungary
In our mouse model, a naturally occuring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (MstnCmpt-dl1Abc) labeled by a tremendous increase in body weight along with signs of muscle weakness and easier fatiguability. To characterize and better understand the mechanism underlying these alterations, we monitored store-operated Ca2+-entry (SOCE) and also measured the cytosolic [Ca2+] (rhod2) using whole cell voltage clamp technique. Since STIM1 and Orai1 endogenous protein levels in the mutant flexor digitorum brevis (FDB) muscles were reduced by ~30% we hypothesized that SOCE, a potential candidate to assist in the longer–term maintenance of EC coupling may be consequently altered. Enzymatically isolated fluo-8 AM loaded FDB fibers were used. To elicit a massive SR Ca2+release a RyR1 agonist (4-chloro-meta-cresol, 4-CmC) was applied in a Ca2+ free medium and in the presence of the SR Ca2+ pump inhibitor (thapsigargin, TG). The above cocktail triggered a deep transient store depletion which in turn switched on SOCE. The peak of this slow Ca2+ transient was normalized to the peak of the SR Ca2+ release transient (0.63±0.09, n=18 vs 0.32±0.08*, n=11, p≤0.02).
3
Ischemia-reperfusion (I/R) injury of the kidney is one of the major causes of acute kidney injury. MicroRNAs are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R-injury. MiR-24 is upregulated in the kidney following I/R-injury of mice (4.7 fold, p
