31936
2023
eng
11
12
article
1
--
2023-05-31
--
Impact of a femoral fracture on outcome after traumatic brain injury — a matched-pair analysis of the TraumaRegister DGU\(^®\)
Traumatic brain injury (TBI) is the leading cause of death and disability in polytrauma and is often accompanied by concomitant injuries. We conducted a retrospective matched-pair analysis of data from a 10-year period from the multicenter database TraumaRegister DGU\(^®\) to analyze the impact of a concomitant femoral fracture on the outcome of TBI patients. A total of 4508 patients with moderate to critical TBI were included and matched by severity of TBI, American Society of Anesthesiologists (ASA) risk classification, initial Glasgow Coma Scale (GCS), age, and sex. Patients who suffered combined TBI and femoral fracture showed increased mortality and worse outcome at the time of discharge, a higher chance of multi-organ failure, and a rate of neurosurgical intervention. Especially those with moderate TBI showed enhanced in-hospital mortality when presenting with a concomitant femoral fracture (p = 0.037). The choice of fracture treatment (damage control orthopedics vs. early total care) did not impact mortality. In summary, patients with combined TBI and femoral fracture have higher mortality, more in-hospital complications, an increased need for neurosurgical intervention, and inferior outcome compared to patients with TBI solely. More investigations are needed to decipher the pathophysiological consequences of a long-bone fracture on the outcome after TBI.
Journal of Clinical Medicine
2077-0383
10.3390/jcm12113802
urn:nbn:de:bvb:20-opus-319363
2023-06-07T11:41:39+00:00
sword
swordwue
attachment; filename=deposit.zip
5b83db31cde282f9e7d4e661c6493487
Journal of Clinical Medicine (2023) 12:11, 3802. https://doi.org/10.3390/jcm12113802
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Mila M. Paul
Hannah J. Mieden
Rolf Lefering
Eva K. Kupczyk
Martin C. Jordan
Fabian Gilbert
Rainer H. Meffert
Anna-Leena Sirén
Stefanie Hoelscher-Doht
eng
uncontrolled
traumatic brain injury
eng
uncontrolled
femoral fracture
eng
uncontrolled
damage control orthopedics
eng
uncontrolled
mortality
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II)
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/31936/jcm-12-03802-v2.pdf
30490
2023
eng
3
24
article
1
--
2023-01-21
--
Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation
Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.
International Journal of Molecular Sciences
1422-0067
10.3390/ijms24032128
urn:nbn:de:bvb:20-opus-304904
2023-03-14T06:02:46+00:00
sword
swordwue
attachment; filename=deposit.zip
9a3b69c8e3fd636316b0ba75befe71f1
International Journal of Molecular Sciences (2023) 24:3, 2128. https://doi.org/10.3390/ijms24032128
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Achmed Mrestani
Katharina Lichter
Anna-Leena Sirén
Manfred Heckmann
Mila M. Paul
Martin Pauli
eng
uncontrolled
active zone
eng
uncontrolled
nanotopology
eng
uncontrolled
neuromuscular junction
eng
uncontrolled
high-pressure freezing/freeze substitution
eng
uncontrolled
PFA in ethanol
eng
uncontrolled
dSTORM
eng
uncontrolled
Drosophila melanogaster
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II)
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/30490/ijms-24-02128.pdf
28822
2022
eng
10
12
article
1
--
2022-09-20
--
Coupling the cardiac voltage-gated sodium channel to channelrhodopsin-2 generates novel optical switches for action potential studies
Voltage-gated sodium (Na\(^+\)) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na\(^+\) influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na\(^+\) channel (Na\(_v\)1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Na\(_v\)1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K\(^+\) channels on the AP shape, we co-expressed either K\(_v\)1.2 or hERG with one of the Na\(_v\)1.5-ChR2 fusions. As expected, both delayed rectifier K\(^+\) channels shortened AP duration significantly. K\(_v\)1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na\(^+\) current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na\(^+\) channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.
Membranes
2077-0375
10.3390/membranes12100907
urn:nbn:de:bvb:20-opus-288228
2022-10-06T13:43:29+00:00
sword
swordwue
attachment; filename=deposit.zip
63392f20baa8116baf99bc6d1bfbee41
Membranes (2022) 12:10, 907. https://doi.org/10.3390/membranes12100907
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Christian vom Dahl
Christoph Emanuel Müller
Xhevat Berisha
Georg Nagel
Thomas Zimmer
eng
uncontrolled
optogenetics
eng
uncontrolled
channelrhodopsin
eng
uncontrolled
voltage-gated Na\(^+\) channel
eng
uncontrolled
action potential
eng
uncontrolled
delayed rectifier potassium channel
eng
uncontrolled
hERG
eng
uncontrolled
long QT syndrome
Biowissenschaften; Biologie
Medizin und Gesundheit
open_access
Physiologisches Institut
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/28822/membranes-12-00907-v2.pdf
28495
2021
eng
9
22
article
1
--
2021-05-03
--
Amelioration of cognitive and behavioral deficits after traumatic brain injury in coagulation factor XII deficient mice
Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII\(^{−/−}\) mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII\(^{−/−}\) mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII\(^{−/−}\) mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII\(^{−/−}\) mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII\(^{−/−}\) mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.
International Journal of Molecular Sciences
1422-0067
10.3390/ijms22094855
urn:nbn:de:bvb:20-opus-284959
2022-09-05T10:33:19+00:00
sword
swordwue
attachment; filename=deposit.zip
aa6ffd9fe51d7d81c85f17133a546f83
International Journal of Molecular Sciences (2021) 22:9, 4855. https://doi.org/10.3390/ijms22094855
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Christian Stetter
Simon Lopez-Caperuchipi
Sarah Hopp-Krämer
Michael Bieber
Christoph Kleinschnitz
Anna-Leena Sirén
Christiane Albert-Weißenberger
eng
uncontrolled
closed head injury
eng
uncontrolled
contact-kinin system
eng
uncontrolled
object recognition memory
eng
uncontrolled
IntelliCage
eng
uncontrolled
Crespi effect
eng
uncontrolled
stress
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Neurologische Klinik und Poliklinik
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/28495/ijms-22-04855-v2.pdf
28578
2021
eng
2
22
article
1
--
2021-01-07
--
Inflammation in the human periodontium induces downregulation of the α\(_1\)- and β\(_1\)-subunits of the sGC in cementoclasts
Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α\(_1\)- and β\(_1\)-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α\(_1\)- and β\(_1\)-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α\(_1\)- and β\(_1\)-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.
International Journal of Molecular Sciences
1422-0067
10.3390/ijms22020539
urn:nbn:de:bvb:20-opus-285783
2022-09-06T17:59:46+00:00
sword
swordwue
attachment; filename=deposit.zip
b5694e9375e03dbe01438f684dc7420a
International Journal of Molecular Sciences (2021) 22:2, 539. https://doi.org/10.3390/ijms22020539
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Yüksel Korkmaz
Behrus Puladi
Kerstin Galler
Peer W. Kämmerer
Agnes Schröder
Lina Gölz
Tim Sparwasser
Wilhelm Bloch
Andreas Friebe
James Deschner
eng
uncontrolled
nitric oxide
eng
uncontrolled
soluble guanylyl cyclase
eng
uncontrolled
cGMP
eng
uncontrolled
cementoclasts
eng
uncontrolled
cementum
eng
uncontrolled
osteoclasts
eng
uncontrolled
alveolar bone
eng
uncontrolled
periodontitis
Medizin und Gesundheit
open_access
Physiologisches Institut
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/28578/ijms-22-00539-v2.pdf
25476
2022
eng
1
12
article
1
--
2022-01-06
--
Characterization and modification of light-sensitive phosphodiesterases from choanoflagellates
Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.
Biomolecules
2218-273X
10.3390/biom12010088
urn:nbn:de:bvb:20-opus-254769
2022-02-05T17:55:47+00:00
sword
swordwue
attachment; filename=deposit.zip
6cd6ea4566baba2f3bc4fdca1496c951
Biomolecules (2022) 12:1, 88. https://doi.org/10.3390/biom12010088
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Yuehui Tian
Shang Yang
Georg Nagel
Shiqiang Gao
eng
uncontrolled
choanoflagellates
eng
uncontrolled
optogenetics
eng
uncontrolled
rhodopsin phosphodiesterase (RhoPDE)
eng
uncontrolled
cGMP
Biowissenschaften; Biologie
open_access
Physiologisches Institut
Import
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/25476/biomolecules-12-00088.pdf
30091
2022
eng
12
40
article
1
--
--
--
Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse
Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{−/−}\) and wild-type mice. In RIM1α\(^{−/−}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0–2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{−/−}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.
Cell Reports
10.1016/j.celrep.2022.111382
urn:nbn:de:bvb:20-opus-300913
@articleLichter.2022, author = Lichter, Katharina and Paul, Mila Marie and Pauli, Martin and Schoch, Susanne and Kollmannsberger, Philip and Stigloher, Christian and Heckmann, Manfred and Sirén, Anna-Leena, year = 2022, title = Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse, pages = 111382, volume = 40, number = 12, journal = Cell reports, doi = 10.1016/j.celrep.2022.111382,
md5:58b8b52b9e7913a27873b5542b8c6815
2023-01-23T07:20:57+00:00
/tmp/phpriU9my
bibtex
63ce355974e956.05775355
Cell Reports (2022) 40:12, 111382. https://doi.org/10.1016/j.celrep.2022.111382
false
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Katharina Lichter
Mila Marie Paul
Martin Pauli
Susanne Schoch
Philip Kollmannsberger
Christian Stigloher
Manfred Heckmann
Anna-Leena Sirén
eng
uncontrolled
active zone
eng
uncontrolled
acute brain slices
eng
uncontrolled
CA3
eng
uncontrolled
electron tomography
eng
uncontrolled
high-pressure freezing
eng
uncontrolled
hippocampal mossy fiber bouton
eng
uncontrolled
RIM1α
eng
uncontrolled
SV pool
eng
uncontrolled
synaptic ultrastructure
eng
uncontrolled
presynaptic
Humanphysiologie
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II)
Theodor-Boveri-Institut für Biowissenschaften
Center for Computational and Theoretical Biology
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/30091/1-s2.0-S2211124722012177-main.pdf
30041
2022
eng
12
article
1
--
--
--
Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap
Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.
Scientific reports
10.1038/s41598-022-06915-z
urn:nbn:de:bvb:20-opus-300411
@articleScherzer.2022, author = Scherzer, Sönke and Huang, Shouguang and Iosip, Anda and Kreuzer, Ines and Yokawa, Ken and Al-Rasheid, Khaled A. S. and Heckmann, Manfred and Hedrich, Rainer, year = 2022, title = Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap, pages = 2851, volume = 12, number = 1, journal = Scientific reports, doi = 10.1038/s41598-022-06915-z,
md5:d404ac54732da953260c61b64caa9578
2023-01-20T07:08:58+00:00
/tmp/phpOy2dZl
bibtex
63ca3e0a953c80.48508011
Scientific Reports (2022) 12:2851. https://doi.org/10.1038/s41598-022-06915-z
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sönke Scherzer
Shouguang Huang
Anda Iosip
Ines Kreuzer
Ken Yokawa
Khaled A. S. Al-Rasheid
Manfred Heckmann
Rainer Hedrich
eng
uncontrolled
biophysics
eng
uncontrolled
drug discovery
eng
uncontrolled
physiology
eng
uncontrolled
plan sciences
Biowissenschaften; Biologie
open_access
Physiologisches Institut
Julius-von-Sachs-Institut für Biowissenschaften
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/30041/s41598-022-06915-z.pdf
29944
2022
eng
16
article
1
--
2022-12-14
--
Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
Introduction
Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.
Methods
We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.
Results and discussion
Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13\(^{GFSTF}\) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13\(^{GFSTF}\), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13\(^{GFSTF}\) subclusters that move toward the AZ center during PHP with unaltered Unc-13\(^{GFSTF}\) protein levels.
Frontiers in Cellular Neuroscience
1662-5102
10.3389/fncel.2022.1074304
urn:nbn:de:bvb:20-opus-299440
2023-01-18T06:19:17+00:00
sword
swordwue
attachment; filename=deposit.zip
b2865a404dcc65a1e7608966b766fbf7
Frontiers in Cellular Neuroscience (2022) 16:1074304. doi:10.3389/fncel.2022.1074304
false
true
Sven Dannhäuser
Achmed Mrestani
Florian Gundelach
Martin Pauli
Fabian Komma
Philip Kollmannsberger
Markus Sauer
Manfred Heckmann
Mila M. Paul
eng
uncontrolled
active zone
eng
uncontrolled
Unc-13
eng
uncontrolled
MiMIC
eng
uncontrolled
presynaptic homeostasis
eng
uncontrolled
nanoarchitecture
eng
uncontrolled
localization microscopy
eng
uncontrolled
STORM
eng
uncontrolled
HDBSCAN
Medizin und Gesundheit
open_access
Physiologisches Institut
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II)
Theodor-Boveri-Institut für Biowissenschaften
Import
Center for Computational and Theoretical Biology
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/29944/fncel-16-1074304.pdf
27468
2022
eng
14
article
1
--
2022-05-18
--
Visualizing presynaptic active zones and synaptic vesicles
The presynaptic active zone (AZ) of chemical synapses is a highly dynamic compartment where synaptic vesicle fusion and neurotransmitter release take place. During evolution the AZ was optimized for speed, accuracy, and reliability of chemical synaptic transmission in combination with miniaturization and plasticity. Single-molecule localization microscopy (SMLM) offers nanometer spatial resolution as well as information about copy number, localization, and orientation of proteins of interest in AZs. This type of imaging allows quantifications of activity dependent AZ reorganizations, e.g., in the context of presynaptic homeostatic potentiation. In combination with high-pressure freezing and optogenetic or electrical stimulation AZs can be imaged with millisecond temporal resolution during synaptic activity. Therefore SMLM allows the determination of key parameters in the complex spatial environment of AZs, necessary for next generation simulations of chemical synapses with realistic protein arrangements.
Frontiers in Synaptic Neuroscience
1663-3563
10.3389/fnsyn.2022.901341
urn:nbn:de:bvb:20-opus-274687
2022-06-01T18:02:48+00:00
sword
swordwue
attachment; filename=deposit.zip
83dcee206ce77645bc810703e3f571a9
Frontiers in Synaptic Neuroscience (2022) 14:901341. DOI:10.3389/fnsyn.2022.901341
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Manfred Heckmann
Martin Pauli
eng
uncontrolled
active zone
eng
uncontrolled
depression
eng
uncontrolled
facilitation
eng
uncontrolled
plasticity
eng
uncontrolled
potentiation
eng
uncontrolled
synapse
Medizin und Gesundheit
open_access
Physiologisches Institut
Import
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/27468/fnsyn-14-901341.pdf
30090
2022
eng
11
article
1
--
--
--
Network instability dynamics drive a transient bursting period in the developing hippocampus in vivo
Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying network burstiness in the intact developing brain are largely unknown. Here, we use two-photon Ca\(^{2+}\) imaging to comprehensively map the developmental trajectories of spontaneous network activity in the hippocampal area CA1 of mice in vivo. We unexpectedly find that network burstiness peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network burstiness reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts (NBs) recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for NB generation. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.
eLife
10.7554/eLife.82756
urn:nbn:de:bvb:20-opus-300906
@articleGraf.2022, author = Graf, Jürgen and Rahmati, Vahid and Majoros, Myrtill and Witte, Otto W. and Geis, Christian and Kiebel, Stefan J. and Holthoff, Knut and Kirmse, Knut, year = 2022, title = Network instability dynamics drive a transient bursting period in the developing hippocampus in vivo, volume = 11, journal = eLife, doi = 10.7554/eLife.82756,
md5:68d15444db1eeb666b827a9d9d78cd69
2023-01-23T07:20:57+00:00
/tmp/phpriU9my
bibtex
63ce355974e956.05775355
eLife 2022, 11:e82756. DOI: 10.7554/eLife.82756
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Jürgen Graf
Vahid Rahmati
Myrtill Majoros
Otto W. Witte
Christian Geis
Stefan J. Kiebel
Knut Holthoff
Knut Kirmse
eng
uncontrolled
hippocampus
eng
uncontrolled
spontaneous network activity
eng
uncontrolled
transient bursting
Humanphysiologie
open_access
Physiologisches Institut
Förderzeitraum 2022
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/30090/elife_Graf.pdf
26851
2021
eng
225-241
2
128
article
1
--
--
--
Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly
Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
Journal of Neural Transmission
1435-1463
10.1007/s00702-021-02303-5
33560471
urn:nbn:de:bvb:20-opus-268519
publish
Journal of Neural Transmission 2021, 128(2):225-241. DOI: 10.1007/s00702-021-02303-5
602805
728018
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Charline Jansch
Georg C. Ziegler
Andrea Forero
Sina Gredy
Sina Wäldchen
Maria Rosaria Vitale
Evgeniy Svirin
Johanna E. M. Zöller
Jonas Waider
Katharina Günther
Frank Edenhofer
Markus Sauer
Erhard Wischmeyer
Klaus-Peter Lesch
eng
uncontrolled
neuropsychiatric disorders
eng
uncontrolled
human induced pluripotent stem cell (hiPSC)
eng
uncontrolled
serotonin-specific neurons
eng
uncontrolled
median and dorsal raphe
eng
uncontrolled
synapse formation
eng
uncontrolled
Cadherin-13 (CDH13)
Medizin und Gesundheit
open_access
Physiologisches Institut
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Theodor-Boveri-Institut für Biowissenschaften
OpenAIRE
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/26851/Jansch_Neural.pdf
26767
2022
eng
181–183
2
474
article
1
--
--
--
Non-linear GABA\(_{A}\) receptors promote synaptic inhibition in developing neurons
No abstract available.
Pflügers Archiv - European Journal of Physiology
1432-2013
10.1007/s00424-021-02652-w
34907453
urn:nbn:de:bvb:20-opus-267674
publish
Pflügers Archiv - European Journal of Physiology 2022, 474(2):181–183. DOI: /10.1007/s00424-021-02652-w
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Knut Kirmse
eng
uncontrolled
synaptic inhibition
eng
uncontrolled
neurons
eng
uncontrolled
GABA\(_{A}\) receptors
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/26767/Kirmse_Pfluegers.pdf
26549
2021
eng
109770
1
37
article
1
2022-04-04
--
--
Active zone compaction correlates with presynaptic homeostatic potentiation
Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.
Cell Reports
10.1016/j.celrep.2021.109770
urn:nbn:de:bvb:20-opus-265497
publish
Cell Reports (2021) 37:1, 109770. https://doi.org/10.1016/j.celrep.2021.109770
false
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Achmed Mrestani
Martin Pauli
Philip Kollmannsberger
Felix Repp
Robert J. Kittel
Jens Eilers
Sören Doose
Markus Sauer
Anna-Leena Sirén
Manfred Heckmann
Mila M. Paul
eng
uncontrolled
active zone
eng
uncontrolled
Bruchpilot
eng
uncontrolled
RIM-binding protein
eng
uncontrolled
compaction
eng
uncontrolled
homeostasis
eng
uncontrolled
presynaptic plasticity
eng
uncontrolled
super-resolution microscopy
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II)
Theodor-Boveri-Institut für Biowissenschaften
Center for Computational and Theoretical Biology
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/26549/1-s2.0-S2211124721012249-main.pdf
25983
2021
eng
407
4
article
1
2022-03-10
--
--
Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections
Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.
Communications Biology
10.1038/s42003-021-01939-z
urn:nbn:de:bvb:20-opus-259830
publish
Communications Biology (2021) 4:407. DOI:10.1038/s42003-021-01939-z
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Martin Pauli
Mila M. Paul
Sven Proppert
Achmed Mrestani
Marzieh Sharifi
Felix Repp
Lydia Kürzinger
Philip Kollmannsberger
Markus Sauer
Manfred Heckmann
Anna-Leena Sirén
eng
uncontrolled
mossy fiber synapses
eng
uncontrolled
CA3 pyrimidal cells
eng
uncontrolled
CA2+ channels
eng
uncontrolled
active zone
eng
uncontrolled
hippocampal
eng
uncontrolled
release
eng
uncontrolled
plasticity
eng
uncontrolled
proteins
eng
uncontrolled
platform
eng
uncontrolled
reveals
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Center for Computational and Theoretical Biology
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/25983/s42003-021-01939-z.pdf
24924
2021
eng
8
article
1
--
2021-11-01
--
Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates
Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.
Frontiers in Molecular Biosciences
2296-889X
10.3389/fmolb.2021.750528
urn:nbn:de:bvb:20-opus-249248
2021-11-15T08:06:20+00:00
sword
swordwue
attachment; filename=deposit.zip
c2869d94ca600a07939c1e28e46718d8
Frontiers in Molecular Biosciences (2021) 8:750528. doi: 10.3389/fmolb.2021.750528
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sabine Panzer
Chong Zhang
Tilen Konte
Celine Bräuer
Anne Diemar
Parathy Yogendran
Jing Yu-Strzelczyk
Georg Nagel
Shiqiang Gao
Ulrich Terpitz
eng
uncontrolled
black yeast
eng
uncontrolled
photoreceptor
eng
uncontrolled
microbial rhodopsins
eng
uncontrolled
optogenetics
eng
uncontrolled
proton channel
eng
uncontrolled
membrane trafficking
eng
uncontrolled
fungal rhodopsins
eng
uncontrolled
Aureobasidium
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Import
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/24924/fmolb-08-750528.pdf
24852
2021
eng
10
11
article
1
--
2021-10-17
--
Genotype- and Age-Dependent Differences in Ultrasound Vocalizations of SPRED2 Mutant Mice Revealed by Machine Deep Learning
Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic–pituitary–adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.
Brain Sciences
2076-3425
10.3390/brainsci11101365
urn:nbn:de:bvb:20-opus-248525
2021-11-05T00:07:26+00:00
sword
swordwue
attachment; filename=deposit.zip
d5bb3c0725291c14d2d7ab842cc2bd28
Brain Sciences 2021, 11(10), 1365; https://doi.org/10.3390/brainsci11101365
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Denis Hepbasli
Sina Gredy
Melanie Ullrich
Amelie Reigl
Marco Abeßer
Thomas Raabe
Kai Schuh
eng
uncontrolled
SPRED
eng
uncontrolled
SPRED2
eng
uncontrolled
mice
eng
uncontrolled
neural networks
eng
uncontrolled
ultrasound vocalizations
eng
uncontrolled
DeepSqueak
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Import
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/24852/brainsci-11-01365-v2.pdf
24273
2021
eng
14
10
article
1
--
2021-07-14
--
5-HTT Deficiency in Male Mice Affects Healing and Behavior after Myocardial Infarction
Anxiety disorders and depression are common comorbidities in cardiac patients. Mice lacking the serotonin transporter (5-HTT) exhibit increased anxiety-like behavior. However, the role of 5-HTT deficiency on cardiac aging, and on healing and remodeling processes after myocardial infarction (MI), remains unclear. Cardiological evaluation of experimentally naïve male mice revealed a mild cardiac dysfunction in ≥4-month-old 5-HTT knockout (−/−) animals. Following induction of chronic cardiac dysfunction (CCD) by MI vs. sham operation 5-HTT−/− mice with infarct sizes >30% experienced 100% mortality, while 50% of 5-HTT+/− and 37% of 5-HTT+/+ animals with large MI survived the 8-week observation period. Surviving (sham and MI < 30%) 5-HTT−/− mutants displayed reduced exploratory activity and increased anxiety-like behavior in different approach-avoidance tasks. However, CCD failed to provoke a depressive-like behavioral response in either 5-Htt genotype. Mechanistic analyses were performed on mice 3 days post-MI. Electrocardiography, histology and FACS of inflammatory cells revealed no abnormalities. However, gene expression of inflammation-related cytokines (TGF-β, TNF-α, IL-6) and MMP-2, a protein involved in the breakdown of extracellular matrix, was significantly increased in 5-HTT−/− mice after MI. This study shows that 5-HTT deficiency leads to age-dependent cardiac dysfunction and disrupted early healing after MI probably due to alterations of inflammatory processes in mice.
Journal of Clinical Medicine
2077-0383
10.3390/jcm10143104
urn:nbn:de:bvb:20-opus-242739
2021-08-01T16:39:17+00:00
sword
swordwue
attachment; filename=deposit.zip
c26d74e7ca9ddb8ea8bed92f5c7da38f
Journal of Clinical Medicine 2021, 10(14), 3104; https://doi.org/10.3390/jcm10143104
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sandy Popp
Angelika Schmitt-Böhrer
Simon Langer
Ulrich Hofmann
Leif Hommers
Kai Schuh
Stefan Frantz
Klaus-Peter Lesch
Anna Frey
eng
uncontrolled
chronic heart failure
eng
uncontrolled
myocardial infarction
eng
uncontrolled
serotonin transporter deficient mice
eng
uncontrolled
anxiety
eng
uncontrolled
depression
eng
uncontrolled
behavior
eng
uncontrolled
inflammation
Medizin und Gesundheit
open_access
Physiologisches Institut
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Medizinische Klinik und Poliklinik I
Import
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/24273/jcm-10-03104-v2.pdf
23661
2021
eng
4
11
article
1
--
2021-04-14
--
Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics
Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.
Membranes
2077-0375
10.3390/membranes11040287
urn:nbn:de:bvb:20-opus-236617
2021-05-03T12:56:08+00:00
sword
swordwue
attachment; filename=deposit.zip
b6a448c3255cd1a55a07b2b84737915c
Membranes 2021, 11(4), 287; https://doi.org/10.3390/membranes11040287
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Yang Zhou
Meiqi Ding
Xiaodong Duan
Kai R. Konrad
Georg Nagel
Shiqiang Gao
eng
uncontrolled
optogenetics
eng
uncontrolled
rhodopsin
eng
uncontrolled
light-sensitive anion channel
eng
uncontrolled
surface potential recording
eng
uncontrolled
pollen tube
Medizin und Gesundheit
open_access
Physiologisches Institut
Julius-von-Sachs-Institut für Biowissenschaften
Import
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23661/membranes-11-00287.pdf
23506
2020
eng
6841–6855
39
article
1
2021-04-21
--
--
The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression
The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
Oncogene
0950-9232
10.1038/s41388-020-01477-8
urn:nbn:de:bvb:20-opus-235064
publish
Oncogene (2020) 39:6841–6855. https://doi.org/10.1038/s41388-020-01477-8
true
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Christina Jessen
Julia K. C. Kreß
Apoorva Baluapuri
Anita Hufnagel
Werner Schmitz
Susanne Kneitz
Sabine Roth
André Marquardt
Silke Appenzeller
Casten P. Ade
Valerie Glutsch
Marion Wobser
José Pedro Friedmann-Angeli
Laura Mosteo
Colin R. Goding
Bastian Schilling
Eva Geissinger
Elmar Wolf
Svenja Meierjohann
eng
uncontrolled
NRF2
eng
uncontrolled
melanoma malignancy
eng
uncontrolled
COX2 expression
Biowissenschaften; Biologie
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie
Medizinische Klinik und Poliklinik II
Rudolf-Virchow-Zentrum
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23506/s41388-020-01477-8.pdf
23620
2020
eng
20
21
article
1
2021-04-29
--
--
Advances, perspectives and potential engineering strategies of light-gated phosphodiesterases for optogenetic applications
The second messengers, cyclic adenosine 3′-5′-monophosphate (cAMP) and cyclic guanosine 3′-5′-monophosphate (cGMP), play important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light, and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. The spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities for spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase genes from protists established a new and direct biological connection between light and PDEs. Light-regulated PDEs are under development, and of demand to complete the toolkit for cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs, and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation.
International Journal of Molecular Sciences
1422-0067
10.3390/ijms21207544
urn:nbn:de:bvb:20-opus-236203
publish
International Journal of Molecular Sciences (2020) 21:20, 7544. https://doi.org/10.3390/ijms21207544
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Yuehui Tian
Shang Yang
Shiqiang Gao
eng
uncontrolled
cyclic nucleotides
eng
uncontrolled
phosphodiesterases (PDEs)
eng
uncontrolled
optogenetics
eng
uncontrolled
cAMP
eng
uncontrolled
cGMP
Medizin und Gesundheit
open_access
Physiologisches Institut
Förderzeitraum 2020
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23620/ijms-21-07544-v2.pdf
23605
2020
eng
5
21
article
1
2021-04-28
--
--
Visualizing the synaptic and cellular ultrastructure in neurons differentiated from human induced neural stem cells - an optimized protocol
The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.
International Journal of Molecular Sciences
10.3390/ijms21051708
urn:nbn:de:bvb:20-opus-236053
1422-0067
publish
International Journal of Molecular Sciences (2020) 21:5, 1708. https://doi.org/10.3390/ijms21051708
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Philipp Capetian
Lorenz Müller
Jens Volkmann
Manfred Heckmann
Süleyman Ergün
Nicole Wagner
eng
uncontrolled
transmission electron microscopy
eng
uncontrolled
human neurons
eng
uncontrolled
induced neural stem cells
eng
uncontrolled
synapse
eng
uncontrolled
synaptic vesicles
eng
uncontrolled
high contrast
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Physiologisches Institut
Neurologische Klinik und Poliklinik
Förderzeitraum 2020
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/23605/ijms-21-01708-v2.pdf
17853
2016
eng
12
11
article
1
2019-03-22
--
--
TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation
The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca\(^{2+}\) component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na\(^{+}\) and Ca\(^{2+}\) influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca\(^{2+}\) influx which has been regarded as ideal Ca\(^{2+}\) source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca\(^{2+}\) signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca\(^{2+}\) transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca\(^{2+}\) cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca\(^{2+}\) transient amplitude at baseline and TRPC4β increasing the Ca\(^{2+}\) peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca\(^{2+}\) influx when treated with Ang II under non-pacing conditions. Consistent with the Ca\(^{2+}\) data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca\(^{2+}\) signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.
PLoS ONE
10.1371/journal.pone.0168446
urn:nbn:de:bvb:20-opus-178539
PLoS ONE 2016, 11(12):e0168446. DOI:10.1371/journal.pone.0168446
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Nadine Kirschmer
Sandra Bandleon
Viktor von Ehrlich-Treuenstätt
Sonja Hartmann
Alice Schaaf
Anna-Karina Lamprecht
Erick Miranda-Laferte
Tanja Langsenlehner
Oliver Ritter
Petra Eder
eng
uncontrolled
Apoptosis
eng
uncontrolled
calcineurin signaling cascade
eng
uncontrolled
small interfering RNAs
eng
uncontrolled
G protein coupled receptors
eng
uncontrolled
hyperexpression techniques
eng
uncontrolled
heart
eng
uncontrolled
adenoviruses
eng
uncontrolled
cardiac pacing
Medizin und Gesundheit
open_access
Physiologisches Institut
Medizinische Klinik und Poliklinik I
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17853/Kirschmer_Plos_One.pdf
20158
2021
eng
doctoralthesis
1
2020-03-13
--
2020-03-09
Function of the Drosophila adhesion-GPCR Latrophilin/CIRL in nociception and neuropathy
Funktionelle Rolle des Drosophila aGPCR Latrophilin/CIRL in Nozizeption und Neuropathie
Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology.
Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family – not only in classical mechanosensory structures.
This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function.
The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons – a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.
Der Tastsinn ist die Fähigkeit, mechanische Reize wahrzunehmen, die für essentielle Verhaltensweisen notwendig sind. Dazu gehören die Vermeidung von Gewebsschädigungen, die Wahrnehmung der Umwelt und soziale Interaktion, aber auch die Propriozeption und das Hören. Daher bleibt die Forschung an Rezeptoren, die mechanische Reize in sensorischen Neuronen in elektrische Signale umwandeln, ein aktueller Forschungsschwerpunk. Die zugrundeliegenden molekularen Mechanismen für die mechanometabotrope Signalübertragung sind trotz der wesentlichen Rolle des Tastsinns in allen Bereichen der Physiologie weitgehend unbekannt.
Adhäsions G-Protein gekoppelte Rezeptoren (aGPCRs), eine große Molekülfamilie mit über 30 Vertretern im Menschen, sind an einer Vielzahl von physiologischen Prozessen beteiligt. Demzufolge wird ein Zusammenhang zwischen diesen Rezeptoren und verschiedenen Erkrankungen des Menschen, wie z. B. Entwicklungsstörungen, Defekte des Nervensystems, Allergien und Krebs, angenommen. Mehrere aGPCRs wurden kürzlich mit mechanosensitiven Funktionen in Verbindung gebracht, was darauf hindeutet, dass die Verarbeitung mechanischer Reize ein gemeinsames Merkmal dieser Rezeptorfamilie ist – nicht nur in klassischen mechanosensorischen Strukturen.
In diesem Projekt wurde Drosophila melanogaster verwendet, um die Funktion des aGPCR-Latrophilin/dCIRL in der mechanischen Nozizeption in vivo zu analysieren. Zu diesem Zweck konzentriert sich diese Arbeit auf mechano-sensorische Neurone (Typ II Klasse IV) der Fruchtfliegenlarve, um die molekularen Mechanismen der dCIRL-Aktivität zu untersuchen. Hierzu wurden noxische mechanische Reize in Kombination mit optogenetischen Werkzeugen, zur Manipulation der Second-Messenger-Signalübertragung, herangezogen.
Zusätzlich wurde ein Neuropathie-Modell etabliert, um eine Beteiligung des aGPCRs dCIRL am beeinträchtigten peripheren Nervensystem zu testen. Zu diesem Zweck untersucht und charakterisiert diese Studie das nozizeptive Verhalten in dCirl-Nullmutanten (dCirlKO) und die RNA-Interferenz (RNAi) Methode, um zellspezifische Manipulationen auszuführen.
Die Ergebnisse zeigen, dass dCirl in spezifischen peripheren sensorischen Neuronen (C4da) transkribiert wird - ein Zelltyp, der Nozizeptoren in Säugern strukturell ähnlich ist und verschiedene nozizeptive sensorische Modalitäten vermittelt.
Darüber hinaus zeigen dCirlKO-Larven ein erhöhtes nozizeptives Verhalten, welches mittels zellspezifischer Reexpression gerettet werden kann. Die Expression von bPAC (bakterielle photoaktivierbare Adenylatcyclase) in diesen nozizeptiven Neuronen ermöglichte es, intrazelluläre Signalkaskaden von CIRL zu untersuchen, welche durch lichtinduzierte Erhöhung von cAMP angeregt werden. Dieser Versuch zeigt, dass dCIRL durch die Modulation nozizeptiver Neuronen eine Herabregulation des nozizeptiven Verhaltens bewirkt.
Angesichts der klinischen Relevanz dieses Ergebnisses wurde die dCirl-Funktion in einem chemisch induzierten Neuropathie-Modell getestet. Dabei stellte sich heraus, dass zellspezifische Überexpression von dCirl eine ausgeprägte Hyperalgesie reduziert, morphologische Schädigungen hingegen nicht gerettet werden konnten.
urn:nbn:de:bvb:20-opus-201580
10.25972/OPUS-20158
University of Leipzig, Faculty of Life Sciences, Institute for Biology
X 129271
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Sven Dannhäuser
deu
swd
Drosophila
deu
swd
Fluoreszenzmikroskopie
deu
swd
Nozizeption
deu
swd
Neuropathie
eng
uncontrolled
nociception
eng
uncontrolled
neuropathy
eng
uncontrolled
adhesion-GPCR
mul
uncontrolled
aGPCR
mul
uncontrolled
dCIRL
mul
uncontrolled
Latrophilin
Physiologie und verwandte Themen
open_access
Graduate School of Life Sciences
Physiologisches Institut
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/20158/Dannhaeuser_Sven_Nociception.pdf
19066
2016
eng
16
2
111
article
1
2019-10-28
--
--
Endothelial actions of atrial natriuretic peptide prevent pulmonary hypertension in mice
The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT(1)-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE-angiotensin pathway in the lung and PH.
Basic Research in Cardiology
10.1007/s00395-016-0541-x
urn:nbn:de:bvb:20-opus-190664
Basic Research in Cardiology (2016) 111:22, 16 Seiten. https://doi.org/10.1007/s00395-016-0541-x
true
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Franziska Werner
Baktybek Kojonazarov
Birgit Gaßner
Marco Abeßer
Kai Schuh
Katharina Völker
Hideo A. Baba
Bhola K. Dahal
Ralph T. Schermuly
Michaela Kuhn
eng
uncontrolled
Atrial natriuretic peptide
eng
uncontrolled
Endothelium
eng
uncontrolled
Guanylyl cyclase-A
eng
uncontrolled
Cyclic GMP
eng
uncontrolled
Pulmonary hypertension
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/19066/Werner_BasicResearchInCardiology_2016.pdf
19282
2020
eng
doctoralthesis
1
2019-12-13
--
2019-12-18
Role of the β subunit of L-type calcium channels in cardiac hypertrophy
Die Rolle der β Untereinheit von L-Typ Kalziumkänalen in der kardialen Hypertrophie
L-type calcium channels (LTCCs) control crucial physiological processes in cardiomyocytes such as the duration and amplitude of action potentials, excitation-contraction coupling and gene expression, by regulating the entry of Ca2+ into the cells. Cardiac LTCCs consist of one pore-forming α1 subunit and the accessory subunits Cavβ, Cavα2δ and Cavγ. Of these auxiliary subunits, Cavβ is the most important regulator of the channel activity; however, it can also have LTCC-independent cellular regulatory functions. Therefore, changes in the expression of Cavβ can lead not only to a dysregulation of LTCC activity, but also to changes in other cellular functions. Cardiac hypertrophy is one of the most relevant risk factors for congestive heart failure and depends on the activation of calcium-dependent prohypertrophic signaling pathways. However, the role of LTCCs and especially Cavβ in this pathology is controversial and needs to be further elucidated.
Of the four Cavβ isoforms, Cavβ2 is the predominant one in cardiomyocytes. Moreover, there are five different splice variants of Cavβ2 (Cavβ2a-e), differing only in the N-terminal region. We reported that Cavβ2b is the predominant variant expressed in the heart. We also revealed that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes. The expression of the nuclear Cavβ2 decreases during in vitro and in vivo induction of cardiomyocyte hypertrophy and overexpression of a nucleus-targeted Cavβ2 completely abolishes the in vitro induced hypertrophy. Additionally, we demonstrated by shRNA-mediated protein knockdown that downregulation of Cavβ2 enhances the hypertrophy induced by the α1-adrenergic agonist phenylephrine (PE) without involvement of LTCC activity. These results suggest that Cavβ2 can regulate cardiac hypertrophy through LTCC-independent pathways. To further validate the role of the nuclear Cavβ2, we performed quantitative proteome analyses of Cavβ2-deficient neonatal rat cardiomyocytes (NRCs). The results show that downregulation of Cavβ2 influences the expression of various proteins, including a decrease of calpastatin, an inhibitor of the calcium-dependent cysteine protease calpain. Moreover, downregulation of Cavβ2 during cardiomyocyte hypertrophy drastically increases calpain activity as compared to controls after treatment with PE. Finally, the inhibition of calpain by calpeptin abolishes the increase in PE-induced hypertrophy in Cavβ2-deficient cells. These results suggest that nuclear Cavβ2 has Ca2+- and LTCC-independent functions during the development of hypertrophy. Overall, our results indicate a new role for Cavβ2 in antihypertrophic signaling in cardiac hypertrophy.
Durch die Regulation des Calciumeintritts in die Zellen kontrollieren L-Typ-Calciumkanäle (LTCCs) wichtige physiologische Prozesse wie die Dauer und Amplitude von Aktionspotentialen, die elektromechanische Kopplung und die Genexpression in Kardiomyozyten. Kardiale LTCCs bestehen aus einer porenformenden α1 Untereinheit und Hilfsuntereinheiten wie Cavβ, Cavα2δ und Cavγ. Von diesen Hilfsuntereinheiten ist Cavβ der wichtigste Regulator der Kanalfunktion, wobei Cavβ auch LTCC-unabhängige zelluläre und regulatorische Funktionen haben kann. Veränderungen in der Expression dieses Proteins können daher zu einer Fehlregulation der LTCC-Aktivität führen, jedoch auch zu Veränderungen von anderen zellulären Funktionen. Einer der häufigsten Risikofaktoren für kongestive Herzinsuffizienz ist die kardiale Hypertrophie, welche abhängig ist von der Aktivierung von Calcium-abhängigen prohypertrophen Signalwegen. Die Rolle von LTCCs und insbesondere von Cavβ in dieser Erkrankung ist jedoch kontrovers und muss noch weiter erforscht werden.
Von den vier Cavβ Splicevarianten ist Cavβ2 die dominierende Form in Kardiomyozyten. Darüber hinaus existieren fünf verschiedene Splicevarianten von Cavβ2 (Cavβ2a-e), die sich jeweils nur in der N-terminalen Region unterscheiden. Wir konnten demonstrieren, dass von diesen Splicevarianten überwiegend Cavβ2b im Herzen exprimiert wird. Außerdem konnten wir zeigen, dass ein Teil von Cavβ2 im Nukleus von Kardiomyozyten zu finden ist. Die Expression von nuklearem Cavβ2 verringert sich während der in vitro und in vivo induzierten kardialen Hypertrophie und außerdem verhindert die Überexpression von im Kern lokalisiertem Cavβ2 die in vitro induzierte Hypertrophie komplett. Zusätzlich konnten wir demonstrieren, dass die Reduktion von Cavβ2 mittels shRNA zu einer Steigerung der Hypertrophie induziert durch die Stimulation mit dem α1-adrenergen Agonisten Phenylephrin (PE) führt, ohne dass die LTCC-Aktivität beteiligt ist. Diese Ergebnisse legen nahe, dass Cavβ2 die Entstehung von Hypertrophie durch LTCC-unabhängige Signalwege beeinflussen kann. Um die Rolle von nuklearem Cavβ2 zu bekräftigen, haben wir quantitative Proteomanalysen von Cavβ2 defizienten neonatalen Rattenkardiomyozyten (NRCs) durchgeführt. Die Ergebnisse zeigen, dass die Reduktion von Cavβ2 die Expression verschiedener Proteine beeinflusst, zum Beispiel wird Calpastatin, ein Inhibitor der calciumabhängigen Cysteinproteasen Calpain, herunterreguliert. Außerdem wird durch die Cavβ2 Reduktion während der Hypertrophie von Kardiomyozyten die Calpainaktivität verglichen mit den Kontrollen signifikant erhöht. Letztendlich konnten wir zeigen, dass die Inhibierung von Calpain durch Calpeptin die gesteigerte PE-induzierte Hypertrophie in Cavβ2-defizienten Zellen verhindert. Diese Ergebnisse lassen eine Calcium- und LTCC-unabhängige Funktion von nuklearem Cavβ2 während der Entwicklung von Hypertrophie, annehmen. Insgesamt deuten unsere Ergebnisse auf eine neue Rolle von Cavβ2 in den antihypertrophen Signalwegen in der kardialen Hypertrophie hin.
urn:nbn:de:bvb:20-opus-192829
10.25972/OPUS-19282
X 129089
CC BY-SA: Creative-Commons-Lizenz: Namensnennung, Weitergabe unter gleichen Bedingungen 4.0 International
Simone Pickel
deu
swd
Herzhypertrophie
deu
swd
Calciumkanal
deu
swd
Herzmuskelzelle
eng
uncontrolled
L-type calcium channels
eng
uncontrolled
Cavβ subunit
eng
uncontrolled
Calpain
eng
uncontrolled
LTCC-independent function of Cavβ
Physiologie und verwandte Themen
open_access
Fakultät für Biologie
Physiologisches Institut
Deutsches Zentrum für Herzinsuffizienz (DZHI)
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/19282/Pickel_Simone_Dissertation.pdf
18729
2016
eng
041802
4
3
article
1
2019-09-13
--
--
Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome
Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
Neurophotonics
10.1117/1.NPh.3.4.041802
urn:nbn:de:bvb:20-opus-187292
Neurophotonics (2016) 3:4, 041802. https://doi.org/10.1117/1.NPh.3.4.041802
false
true
Sebastian Matthias Markert
Sebastian Britz
Sven Proppert
Marietta Lang
Daniel Witvliet
Ben Mulcahy
Markus Sauer
Mei Zhen
Jean-Louis Bessereau
Christian Stigloher
eng
uncontrolled
caenorhabditis elegans
eng
uncontrolled
localization micoscopy
eng
uncontrolled
fluorescent-probes
eng
uncontrolled
junction proteins
eng
uncontrolled
resolution limit
eng
uncontrolled
direct stochasticoptical reconstruction microscopy
eng
uncontrolled
structured illumination microscopy
eng
uncontrolled
correlative light and electron microscopy
eng
uncontrolled
gap junction
eng
uncontrolled
neural circuits
eng
uncontrolled
nervous-system
eng
uncontrolled
image data
eng
uncontrolled
reconstruction
eng
uncontrolled
innexins
eng
uncontrolled
super-resolution microscopy
Biowissenschaften; Biologie
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/18729/Markert_Neurophotonics_2016.pdf
17052
2017
eng
e28360
6
article
1
2018-10-29
--
--
Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons
Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
eLife
10.7554/eLife.28360
urn:nbn:de:bvb:20-opus-170520
eLife 2017, 6:e28360. DOI: 10.7554/eLife.28360
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Nicole Scholz
Chonglin Guan
Matthias Nieberler
Alexander Grotmeyer
Isabella Maiellaro
Shiqiang Gao
Sebastian Beck
Matthias Pawlak
Markus Sauer
Esther Asan
Sven Rothemund
Jana Winkler
Simone Prömel
Georg Nagel
Tobias Langenhan
Robert J Kittel
eng
uncontrolled
Latrophilin
eng
uncontrolled
adhesion GPCR
eng
uncontrolled
dCIRL
eng
uncontrolled
sensory physiology
eng
uncontrolled
metabotropic signalling
eng
uncontrolled
mechanotransduction
Medizin und Gesundheit
open_access
Institut für Pharmakologie und Toxikologie
Institut für Anatomie und Zellbiologie
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Rudolf-Virchow-Zentrum
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17052/044_Scholz_ELIFE.pdf
17752
2018
eng
643
12
article
1
2019-02-27
--
--
Synthetic light-activated ion channels for optogenetic activation and inhibition
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
Frontiers in Neuroscience
10.3389/fnins.2018.00643
urn:nbn:de:bvb:20-opus-177520
Frontiers in Neuroscience 2018, Volume 12, Article 643. DOI: 10.3389/fnins.2018.00643
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sebastian Beck
Jing Yu-Strzelczyk
Dennis Pauls
Oana M. Constantin
Christine E. Gee
Nadine Ehmann
Robert J. Kittel
Georg Nagel
Shiqiang Gao
eng
uncontrolled
optogenetics
eng
uncontrolled
calcium
eng
uncontrolled
potassium
eng
uncontrolled
bPAC
eng
uncontrolled
CNG channel
eng
uncontrolled
cAMP
eng
uncontrolled
Drosophila melanogaster motoneuron
eng
uncontrolled
rat hippocampal neurons
Biowissenschaften; Biologie
open_access
Physiologisches Institut
Julius-von-Sachs-Institut für Biowissenschaften
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2018
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17752/Beck_Frontiers_in_Neuroscience.pdf
17879
2019
eng
doctoralthesis
1
2019-04-01
--
2019-03-15
Characterisation and application of new optogenetic tools in \(Drosophila\) \(melanogaster\)
Charakterisierung und Anwendung neuer optogenetischer Werkzeuge in \(Drosophila\) \(melanogaster\)
Since Channelrhodopsins has been described first and introduced successfully in freely moving animals (Nagel et al., 2003 and 2005), tremendous impact has been made in this interesting field of neuroscience. Subsequently, many different optogenetic tools have been described and used to address long-lasting scientific issues. Furthermore, beside the ‘classical’ Channelrhodopsin-2 (ChR2), basically a cation-selective ion channel, also altered ChR2 descendants, anion selective channels and light-sensitive metabotropic proteins have expanded the optogenetic toolbox. However, in spite of this variety of different tools most researches still pick Channelrhodopsin-2 for their optogenetic approaches due to its well-known kinetics. In this thesis, an improved Channelrhodopsin, Channelrhodopsin2-XXM (ChR2XXM), is described, which might become an useful tool to provide ambitious neuroscientific approaches by dint of its characteristics. Here, ChR2XXM was chosen to investigate the functional consequences of Drosophila larvae lacking latrophilin in their chordotonal organs. Finally, the functionality of GtACR, was checked at the Drosophila NMJ. For a in-depth characterisation, electrophysiology along with behavioural setups was employed. In detail, ChR2XXM was found to have a better cellular expression pattern, high spatiotemporal precision, substantial increased light sensitivity and improved affinity to its chromophore retinal, as compared to ChR2. Employing ChR2XXM, effects of latrophilin (dCIRL) on signal transmission in the chordotonal organ could be clarified with a minimum of side effects, e.g. possible heat response of the chordotonal organ, due to high light sensitivity. Moreover, optogenetic activation of the chordotonal organ, in vivo, led to behavioural changes. Additionally, GtACR1 was found to be effective to inhibit motoneuronal excitation but is accompanied by unexpected side effects. These results demonstrate that further improvement and research of optogenetic tools is highly valuable and required to enable researchers to choose the best fitting optogenetic tool to address their scientific questions.
Seit dem Channelrhodopsine das erste Mal beschrieben und erfolgreich in lebende Tiere eingebracht wurden (Nagel et al., 2003 und 2005), kam es zu einem beträchtlichen Fortschritt in diesem interessanten Gebiet der Neurowissenschaften. In der nachfolgenden Zeit wurden viele verschiedene optogenetische Werkzeuge beschrieben und zur Bearbeitung neurowissenschaftlicher Fragestellungen angewandt. Des Weiteren haben neben dem „klassischen“ Channelrhodopsin-2 (ChR2), ein im Wesentlichen Kation selektiver Kanal, auch modifizierte ChR2 Abkömmlinge, Anion selektive Kanäle und Licht sensitive metabotrope Proteine, die opotogenetische Werkzeugkiste erweitert. Dennoch greifen die meisten Wissenschaftler trotz der Vielfalt an optogenetischen Werkzeugen meist noch zu Channelrhodopsin-2, da seine Wirkungseigenschaften sehr gut erforscht sind.
In der nachfolgenden Arbeit wird ein weiterentwickeltes Channelrhodopsin, Channelrhodopsin2-XXM (ChR2XXM), beschrieben. Aufgrund seiner vielfältigen Eigenschaften stellt es ein vielversprechendes Werkzeug dar, vor allem für zukünftige neurowissenschaftliche Forschungsarbeiten. Hierbei wurde ChR2XXM eingesetzt, um zu untersuchen welche Auswirkungen das Fehlen von Latrophilin im Chordotonal Organ von Drosophilalarven hat. Schließlich wurde noch die Funktionalität von GtACR an der neuromuskulären Endplatte der Drosophila überprüft. Für die umfassende Charakterisierung wurden elektrophysiologische und verhaltensbasierte Experimente an Larven durchgeführt. Es konnte gezeigt werden, dass ChR2XXM aufgrund einer erhöhten Affinität zu dem Chromophore Retinal, im Vergleich zu ChR2 ein besseres zelluläres Expressionsmuster, eine bessere zeitliche Auflösung und eine erheblich höhere Lichtsensitiviät aufweist. Durch den Einsatz von ChR2XXM konnte, aufgrund der hohen Lichtsensitiviät, mit nur minimalen Nebeneffekten, wie z.B. mögliche Wärmeaktivierung des Chordotonalorgans, der Einfluss von Latrophilin (dCIRL) auf die Signaltransmission im Chordotonalorgan, aufgeklärt werden. Ferner führte eine optogenetische, in vivo, Aktivierung des Chordotonalorgans zu Verhaltensänderungen. Zusätzlich konnte gezeigt werden, dass GtACR1 zwar effektiv motoneuronale Erregung inhibieren kann, dies aber von unerwarteten Nebeneffekten begleitet wird. Diese Ergebnisse zeigen auf, dass weitere Forschung und Verbesserungen im Bereich der optogenetischen Werkzeuge sehr wertvoll und notwendig ist, um Wissenschaftlern zu erlauben das am besten geeignetste optogenetische Werkzeug für ihre wissenschaftlichen Fragestellungen auswählen zu können.
urn:nbn:de:bvb:20-opus-178793
10.25972/OPUS-17879
X 128134
CC BY-NC-SA: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Weitergabe unter gleichen Bedingungen 4.0 International
Alexander Grotemeyer
deu
swd
Optogenetik
deu
swd
Taufliege
deu
swd
Elektrophysiologie
deu
swd
Channelrhodopsin-2
eng
uncontrolled
optogenetics
eng
uncontrolled
Drosophila melanogaster
eng
uncontrolled
Channelrhodopsin
eng
uncontrolled
Electrophysiology
Humanphysiologie
open_access
Physiologisches Institut
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17879/Grotemeyer_Alexander_Dissertation.pdf
17665
2018
eng
136-140
28
article
1
2019-02-14
--
--
Generation of a human induced pluripotent stem cell (iPSC) line from a 51-year-old female with attention-deficit/hyperactivity disorder (ADHD) carrying a duplication of SLC2A3
Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype. This in vitro cellular model can be used to study the role of risk genes in the pathogenesis of ADHD, in a patient-specific manner.
Stem Cell Research
10.1016/j.scr.2018.02.005
urn:nbn:de:bvb:20-opus-176654
Stem Cell Research 2018, 28, 136-140. DOI: 10.1016/j.scr.2018.02.005
602805
728018
false
true
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Charline Jansch
Katharina Günther
Jonas Waider
Georg C. Ziegler
Andrea Forero
Sina Kollert
Evgeniy Svirin
Dirk Pühringer
Chee Keong Kwok
Reinhard Ullmann
Anna Maierhofer
Julia Flunkert
Thomas Haaf
Frank Edenhofer
Klaus-Peter Lesch
eng
uncontrolled
ADHD
eng
uncontrolled
SLC2A3
eng
uncontrolled
induced pluripotent stem cells
Menschliche Anatomie, Zytologie, Histologie
open_access
Institut für Humangenetik
Institut für Anatomie und Zellbiologie
Physiologisches Institut
Lehrstuhl für Molekulare Psychiatrie
OpenAIRE
Förderzeitraum 2018
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17665/Jansch_Stem_Cell_Research.pdf
17632
2018
eng
103
17
article
1
2019-02-11
--
--
β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice
Background:
The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
Methods:
Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in β-cells (β GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. β-cell size and number were measured by immunofluorescence-based islet morphometry.
Results:
In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from β GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in β GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in β-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and β-cell morphology were similar in β GC-A KO mice and control littermates. However, HFD-fed β GC-A KO animals had accelerated glucose intolerance and diminished adaptative β-cell proliferation.
Conclusions:
Our studies of β GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate β-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of β-cells to HFD-induced obesity. Impaired β-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.
Cardiovascular Diabetology
10.1186/s12933-018-0747-3
urn:nbn:de:bvb:20-opus-176322
Cardiovascular Diabetology (2018) 17:103. DOI: 10.1186/s12933-018-0747-3
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sabine Tauscher
Hitoshi Nakagawa
Katharina Völker
Franziska Werner
Lisa Krebes
Tamara Potapenko
Sören Doose
Andreas L. Birkenfeld
Hideo A. Baba
Michaela Kuhn
eng
uncontrolled
cylic GMP
eng
uncontrolled
guanylyl cyclase-A
eng
uncontrolled
insulin
eng
uncontrolled
natriuretic peptides
eng
uncontrolled
obesity
eng
uncontrolled
β-cells
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2018
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17632/Tauscher_Cardiovascular_Diabetology.pdf
14899
2015
eng
7
9
article
1
2017-05-22
--
--
Super-resolution microscopy of the synaptic active zone
Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins
Frontiers in Cellular Neuroscience
10.3389/fncel.2015.00007
urn:nbn:de:bvb:20-opus-148997
Frontiers in Cellular Neuroscience 9:7 (2015). DOI: 10.3389/fncel.2015.00007
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Nadine Ehmann
Markus Sauer
Robert J. Kittel
eng
uncontrolled
excitation-secretion coupling
eng
uncontrolled
Ca\(^{2+}\) channels
eng
uncontrolled
structure-function relationships
eng
uncontrolled
super-resolution microscopy
eng
uncontrolled
active zone
eng
uncontrolled
presynaptic calcium
eng
uncontrolled
neurotransmitter release
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14899/053_Ehmann_Frontiers_in_Cellular_Neuroscience.pdf
14898
2015
eng
29
9
article
1
2017-05-22
--
--
Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp\(^{69}\)). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp\(^{69}\) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt\(^{KD}\)) in wildtype (wt), brp\(^{69}\) and rab3 null mutants (rab3\(^{rup}\)), where Brp is concentrated at a small number of AZs. At wt and rab3\(^{rup}\) synapses, syt\(^{KD}\) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt\(^{KD}\) did not alter EPSC amplitude at brp\(^{69}\) synapses, but shortened delay and rise time. In fact, following syt\(^{KD}\), these kinetic properties were strikingly similar in wt and brp\(^{69}\), which supports the notion that Syt protracts release at brp\(^{69}\) synapses. To gain insight into this surprising role of Syt at brp\(^{69}\) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At tonic type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt\(^{KD}\). Notably, syt\(^{KD}\) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt\(^{KD}\) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
Frontiers in Cellular Neuroscience
10.3389/fncel.2015.00029
urn:nbn:de:bvb:20-opus-148988
Frontiers in Cellular Neuroscience 9:29 (2015). DOI: 10.3389/fncel.2015.00029
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Mila M. Paul
Martin Pauli
Nadine Ehmann
Stefan Hallermann
Markus Sauer
Robert J. Kittel
Manfred Heckmann
eng
uncontrolled
neuromuscular junction
eng
uncontrolled
Bruchpilot
eng
uncontrolled
synaptic delay
eng
uncontrolled
dSTORM
eng
uncontrolled
synaptotagmin
eng
uncontrolled
presynaptic differentiation
eng
uncontrolled
neurotransmitter release
eng
uncontrolled
active zone
eng
uncontrolled
synaptic transmission
eng
uncontrolled
fluorescent probes
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14898/052_Paul_Frontiers_in_Cellular_Neuroscience.pdf
14862
2015
eng
866-874
11
article
1
2017-05-17
--
--
The adhesion GPCR Latrophilin/CIRL shapes mechanosensation
G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. Our study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. We show that dCirl shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. Our results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily.
Cell Reports
10.1016/j.celrep.2015.04.008
urn:nbn:de:bvb:20-opus-148626
Cell Reports 2015, 11, 866-874. DOI: 10.1016/j.celrep.2015.04.008
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Nicole Scholz
Jennifer Gehring
Chonglin Guan
Dmitrij Ljaschenko
Robin Fischer
Vetrivel Lakshmanan
Robert J. Kittel
Tobias Langenhan
eng
uncontrolled
\(\alpha\)-latrotoxin
eng
uncontrolled
chordotonal organs
eng
uncontrolled
Johnstons organ
eng
uncontrolled
ligand CD55
eng
uncontrolled
hearing
eng
uncontrolled
binding
eng
uncontrolled
shear stress
eng
uncontrolled
protein-coupled receptors
eng
uncontrolled
drosophila larvae
eng
uncontrolled
domain
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14862/032_Scholz_Cell_Reports.pdf
14831
2015
eng
12548
5
article
1
2017-05-10
--
--
Activation of TRESK channels by the inflammatory mediator lysophosphatidic acid balances nociceptive signalling
In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IK\(_{SO}\). A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K\(^{+}\) currents upon LPA application. In DRG neurons nociception can result from TRPV\(_{1}\) activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV\(_{1}\) and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IK\(_{SO}\) after application of LPA whereas under these conditions IK\(_{SO}\) in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders.
Scientific Reports
10.1038/srep12548
urn:nbn:de:bvb:20-opus-148312
Scientific Reports 5:12548 (2015). DOI: 10.1038/srep12548
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sina Kollert
Benjamin Dombert
Frank Döring
Erhard Wischmeyer
eng
uncontrolled
protein coupled receptors
eng
uncontrolled
molecular mechanisms
eng
uncontrolled
neuropathic pain
eng
uncontrolled
migraine
eng
uncontrolled
initiation
eng
uncontrolled
modulation
eng
uncontrolled
cells
eng
uncontrolled
sensory neurons
eng
uncontrolled
domain K\(^{+}\) channels
eng
uncontrolled
2-pore potassium channel
Medizin und Gesundheit
open_access
Institut für Klinische Neurobiologie
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14831/013_Kollert_Scientific_Reports.pdf
14518
2015
eng
1389-1400
8
article
1
2017-03-02
--
--
Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.
Disease Models & Mechanisms
10.1242/dmm.021246
urn:nbn:de:bvb:20-opus-145185
Disease Models & Mechanisms (2015) 8, 1389-1400. DOI: 10.1242/dmm.021246
Katherina Beck
Nadine Ehmann
Till F. M. Andlauer
Dmitrij Ljaschenko
Katrin Strecker
Matthias Fischer
Robert J. Kittel
Thomas Raabe
eng
uncontrolled
mrsk2 KO mouse
eng
uncontrolled
S6KII RSK
eng
uncontrolled
transmission
eng
uncontrolled
neuromuscular junction
eng
uncontrolled
synapse
eng
uncontrolled
MAPK signaling
eng
uncontrolled
axonal transport
eng
uncontrolled
motoneuron
eng
uncontrolled
RSK
eng
uncontrolled
Drosophila
eng
uncontrolled
mechanisms
eng
uncontrolled
plasticity
eng
uncontrolled
protein kinase
eng
uncontrolled
signal transduction pathway
eng
uncontrolled
mitochondrial transport
eng
uncontrolled
glutamate receptor
Medizin und Gesundheit
open_access
Physiologisches Institut
Institut für Medizinische Strahlenkunde und Zellforschung
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Rudolf-Virchow-Zentrum
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14518/095_Beck_Disease_Models_Mechanisms.pdf
16232
2016
eng
1238-1246
5
17
article
1
2018-05-29
--
--
cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons
The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.
Cell Reports
10.1016/j.celrep.2016.09.090
urn:nbn:de:bvb:20-opus-162324
Cell Reports 17, 1238–1246 doi:10.1016/j.celrep.2016.09.090
324612
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Isabella Maiellaro
Martin J. Lohse
Robert J. Kitte
Davide Calebiro
eng
uncontrolled
cAMP
eng
uncontrolled
synaptic plasticity
eng
uncontrolled
PDE
eng
uncontrolled
octopamine
eng
uncontrolled
FRET
eng
uncontrolled
active zone
eng
uncontrolled
dunce
eng
uncontrolled
GPCR
Humanphysiologie
open_access
Institut für Pharmakologie und Toxikologie
Physiologisches Institut
Rudolf-Virchow-Zentrum
OpenAIRE
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/16232/Maiellaro_CellReports_2016.pdf
15448
2017
eng
doctoralthesis
1
2017-11-03
--
2017-10-17
SH3-mediated protein interactions of Mena and VASP
SH3 vermittelte Proteininteraktionen von Mena und VASP
Regulation of actin cytoskeletal turnover is necessary to coordinate cell movement and cell adhesion. Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family are important mediators in cytoskeleton control, linking cyclic nucleotide signaling pathways to actin assembly. In mammals, the Ena/VASP family consists of mammalian Enabled (Mena), VASP, and Ena-VASP-like (EVL). The family members share a tripartite domain organization, consisting of an N-terminal Ena/VASP homology 1 (EVH1) domain, a central proline-rich region (PRR), and a C-terminal EVH2 domain. The EVH1 domain mediates binding to the focal adhesion proteins vinculin and zyxin, the PRR interacts with the actin-binding protein profilin and with Src homology 3 (SH3) domains, and the EVH2 domain mediates tetramerization and actin binding.
Endothelial cells line vessel walls and form a semipermeable barrier between blood and the underlying tissue. Endothelial barrier function depends on the integrity of cell-cell junctions and defective sealing of cell-cell contacts results in vascular leakage and edema formation. In a previous study, we could identify a novel interaction of the PRR of VASP with αII-spectrin. VASP-targeting to endothelial cell-cell contacts by interaction with the αII-spectrin SH3 domain is sufficient to initiate perijunctional actin filament assembly, which in turn stabilizes cell-cell contacts and decreases endothelial permeability. Conversely, barrier function of VASP-deficient endothelial cells and microvessels of VASP- null mice is defective, demonstrating that αII-spectrin/VASP complexes regulate endothelial barrier function in vivo.
The aim of the present study was to characterize the structural aspects of the binding of Ena/VASP proteins to αII-spectrin in more detail. These data are highly relevant to understand the cardiovascular function of VASP and its subcellular targeting. In the present study, the following points were experimentally addressed:
1. Comparison of the interaction between αII-spectrin and Mena, VASP, or EVL
In contrast to the highly conserved EVH1/EVH2 domains, the PRR is the most divergent part within the Ena/VASP proteins and may differ in binding modes and mechanisms of regulation. More specifically, VASP contains a triple GP5 motif, whereas EVL and Mena contain one or more GP6 motifs or even longer proline stretches. In the present study, we used peptide scans and competitive αII-spectrin SH3 pull-down assays with the recombinant Mena, VASP, and VASP mutants to investigate the relative binding efficiency. Our results indicate that binding of the αII-spectrin SH3 domain to GP6 motifs is superior to GP5 motifs, giving a rationale for a stronger interaction of αII-spectrin with EVL and Mena than with VASP.
2. Interaction of SH3i with Ena/VASP proteins
In the mammalian heart, an αII-spectrin splice variant exists (SH3i), which contains a 20 amino acid insertion C-terminal to the SH3 domain. We used GST-fusion proteins of αII-spectrin, comprising the SH3 domain with or without the alternatively spliced amino acids, to pull-down recombinant Mena, VASP or VASP mutants. The results demonstrate a substantially increased binding of the C-terminal extended SH3 domain as compared to the general αII-spectrin isoform without the 20 amino acid insertion. These findings were also confirmed in pull-down experiments with heart lysates and purified Mena from heart muscle. The increased binding was not due to an alternative, SH3-independent binding interface because a pointmutation of the SH3 domain (W1004R) in the alternatively spliced αII-spectrin isoform completely abrogated the interaction. To analyze the interaction of SH3i and Ena/VASP proteins in living cells, we expressed the extended SH3 domain as GFP fusion proteins in endothelial cells. Here, we observed an extensive co-localization with Mena and VASP at the leading edge of lamellipodia confirming the in vivo relevance of the interaction with potential impact on cell migration and angiogenesis.
3. Binding affinity and influence of the Ena/VASP tetramerization domain
We also determined the binding affinity of the general and the alternatively spliced αII-spectrin SH3 with Ena/VASP proteins by isothermal titration calorimetry (ITC) using a peptide from the PRR of Mena (collaboration with Dr. Stephan Feller, University of Oxford). Surprisingly, the binding affinity of the general SH3 domain was low (~900 μM) as compared to other SH3 domain- mediated interactions, which commonly display binding constants in the low micromolar range. Furthermore and in contrast to the pull-down assays, we could not detect an increased binding affinity of the C-terminally extended SH3 domain. This could be either explained by the existence of a third protein, which “bridges” the Mena/αII-spectrin complex in the pull-down assays, or, more likely, by the small size of the Mena peptide, which lacks major parts of the Mena protein, including the tetramerization domain. Indeed, it has been previously shown that the tetramerization of Ena is crucial for the interaction with the Abl- SH3 domain, although no SH3 binding sites are found in the tetramerization domain. To address this point experimentally, we used a VASP mutant that lacks the tetramerization domain in pull-down assays. Neither the general nor the alternatively spliced SH3 domain bound to the monomeric VASP, demonstrating the crucial (indirect) impact of Ena/VASP tetramerization on the interaction with αII-spectrin.
In summary, we conclude that the αII-spectrin SH3 domain binds to the proline- rich region of all Ena/VASP proteins. However, binding to EVL and Mena, which both possess one or more GP6 motifs, is substantially more efficient than VASP, which only contains GP5 motifs. The C-terminally extended SH3 domain, which is present in the αII-spectrin splice variant SH3i, binds stronger to the Ena/VASP proteins than the general isoform and expression of the isolated domain is sufficient for co-localization with Ena/VASP in living endothelial cells. Finally, the tetramerization of the Ena/VASP proteins is indispensable for the interaction with either isoform of αII-spectrin.
Die Regulation des Umbaus des Aktinzytoskeletts ist für die Fortbewegung sowie die Adhäsion von Zellen essentiell. Proteine der Enabled/vasodilator- stimulated phosphoprotein (Ena/VASP) Familie sind wichtige Mediatoren bei diesem Prozess, indem sie zyklische Nukleotidprotein-Signalwege mit dem Aktinzytoskelett-Aufbau verknüpfen. In Säugern besteht die Ena/VASP-Protein Familie aus mammalian Enabled (Mena), VASP und Ena-VASP-like (EVL). Diese Proteine teilen sich einen gemeinsamen strukturellen Aufbau: N-terminal befindet sich die Ena/VASP homology 1 (EVH1) Domäne, zentral liegt eine prolinreiche Region (PRR) und C-terminal befindet sich eine EVH2 Domäne. Die EVH1 Domäne vermittelt eine Interaktion mit den fokalen Adhäsionsproteinen Vinculin und Zyxin, die PRR interagiert mit dem aktinbindenden Protein Profilin sowie mit Src homology 3 (SH3) Domänen und die EVH2 Domäne vermittelt die Tetramerisierung der Proteine sowie die Interaktion mit Aktin.
Endothelzellen kleiden die Gefäßwand aus und bilden eine semipermeable Barriere zwischen Blut und dem umgebenden Gewebe. Die Funktion des Endothels hängt dabei von der Integrität der Zell-Zell-Kontakte ab. Die Zerstörung dieser Kontakte führt zu vaskulärer Leckage sowie zur Ausbildung von Ödemen. In einer vorausgehenden Arbeit konnten wir eine neue Interaktion zwischen der PRR von VASP und αII-Spektrin zeigen. Durch die Interaktion mit der SH3 Domäne von αII-Spektrin gelangt VASP an Zell-Zell-Kontakte von Endothelzellen und ist dort in der Lage die Aktinverknüpfung in der Umgebung der Zell-Zell-Kontakte zu initiieren, was wiederum die Zell-Zell-Kontakte stabilisiert und die vaskuläre Permeabilität reduziert. Umgekehrt konnten wir beobachten, dass die Barrierefunktion von Endothelzellen und Mikrogefäßen von VASP-defizienten Mäusen gestört ist, was darauf hindeutet, dass VASP/αII- Spektrin-Komplexe an der Regulation der endothelialen Barrierefunktion in vivo beteiligt sind.
Das Ziel dieser Arbeit war die detaillierte Charakterisierung der Strukturen von Ena/VASP Proteinen und αII-Spektrin, die an der Interaktion zwischen diesen Proteinen beteiligt sind. Diese Daten sind äußerst wichtig um die genaue Funktion von VASP im kardiovaskulären System zu verstehen. In dieser Arbeit wurden die folgenden Punkte genauer analysiert:
1. Vergleich der Interaktion zwischen αII-Spektrin und Mena, VASP oder EVL
Verglichen mit den hochkonservierten EVH1/2 Domänen besitzt die zentrale PRR der Ena/VASP Proteinfamilie die größte Diversität und könnte sich bezüglich der Binde- und Regulationsmechanismen zwischen den einzelnen Proteinen unterscheiden. Im Detail besitzt VASP ein sich dreifach wiederholendes GP5 Motiv wohingegen Mena und EVL ein sich ein- oder mehrfach wiederholendes GP6 Motiv oder noch längere Prolinsequenzen aufweisen. In dieser Arbeit nutzten wir peptide scan arrays und kompetitive αII- Spektrin SH3 pull-down Versuche mit rekombinantem Mena, VASP und VASP Mutanten um die relative Bindungsstärke der Interaktion zu untersuchen. Die Ergebnisse zeigten, dass die Bindung der αII-Spektrin SH3 Domäne an GP6 Motive der Bindung an GP5 Motive überlegen ist, was darauf hindeutet, dass die Interaktion zwischen αII-Spektrin und EVL und Mena stärker ist als die Interaktion mit VASP.
2. Interaktion von SH3i mit Ena/VASP Proteinen
Im Säugerherzen kommt eine αII-Spektrin Splicevariante (SH3i) vor, die eine Insertion aus 20 Aminosäuren C-terminal der SH3 Domäne enthält. Wir nutzten GST-αII-Spektrin Fusionsproteine, die die SH3 Domäne mit oder ohne Aminosäuren-Insertion beinhalteten, im pull-down Versuch mit rekombinantem Mena, VASP oder mit VASP Mutanten. Die Ergebnisse zeigten eine deutlich ansteigende Bindungsrate bei der C-terminal verlängerten SH3 Domäne verglichen zu der üblichen αII-Spektrin Isoform ohne Aminosäureninsertion. Diese Ergebnisse konnten auch durch pull-down Versuche mit Herzlysat und aufgereinigtem Mena aus Herzmuskulatur bestätigt werden. Dieser Effekt auf die Bindung war dabei nicht bedingt durch ein alternatives, SH3-unabhängiges Bindungsmuster, da eine Punktmutation (W1004R) in der Sequenz der SH3 Domäne der alternativ gespleißten αII-Spektrin Isoform die Interaktion komplett aufhob. Um die Interaktion von SH3i und Ena/VASP Proteinen in lebenden Zellen zu untersuchen, exprimierten wir die verlängerte SH3 Domäne als GFP- Fusionsprotein in Endothelzellen. Hierbei konnten wir eine ausgeprägte Kolokalisation mit Mena und VASP an Lamellipodien beobachten, was die Relevanz der Interaktion im lebenden Organismus bestätigt und auf einen potentiellen Effekt auf Zellmigration und Angiogenese schließen lässt.
3. Bindungsaffinität und Einfluss der Ena/VASP Tetramerisierungsdomäne
Zusätzlich wurde die Bindungsaffinität der üblichen sowie der alternativ gespleißten αII-Spektrin SH3 Domäne mit den Ena/VASP Proteinen mittels isothermal titration calorimetry (ITC) bestimmt, wobei ein Peptid bestehend aus der PRR von Mena genutzt wurde (Kollaboration mit Dr. Stephan Feller, Universität von Oxford). Überraschenderweise war die Bindungsaffinität der üblichen SH3 Domäne verglichen mit anderen SH3 Interaktionen, die normalerweise Bindungskonstanten im unteren mikromolaren Bereich aufweisen, niedrig (~900 μM). Außerdem und im Kontrast zu den Ergebnissen der pull-down Versuche konnten wir keine gesteigerte Bindungsaffinität der C- terminal verlängerten SH3 Domäne feststellen. Dies könnte entweder durch die Beteiligung eines dritten Proteins erklärt werden, das den Mena/αII-Spektrin- Komplex in den pull-down Versuchen überbrückt, oder aber, was wahrscheinlicher ist, durch die geringe Größe des Mena Peptids, dem wichtige Bestandteile des Mena Proteins inklusive der Tetramerisierungsdomäne fehlen. Tatsächlich wurde erst kürzlich gezeigt, dass die Tetramerisierung von Ena essentiell für eine Interaktion mit der Abl SH3 Domäne ist, obwohl keine SH3 Bindestellen in der Tetramerisierungsdomäne gefunden wurden. Um dieser Beobachtung nachzugehen, nutzten wir eine VASP Mutante in pull-down Versuchen, der die Tetramerisierungsdomäne fehlt. Weder die übliche noch die alternativ gespleißte SH3 Domäne interagierte mit dem monomeren VASP, was einen essentiellen (indirekten) Einfluss der Tetramerisierung von Ena/VASP Proteinen auf die Interaktion mit αII-Spektrin nahelegt.
Zusammengefasst können wir sagen, dass die αII-Spektrin SH3 Domäne an die prolinreiche Region aller Ena/VASP Proteine bindet. Jedoch ist festzuhalten, dass die Bindung an EVL und Mena, die beide ein oder mehrere GP6 Motive enthalten, deutlich effizienter ist als die Bindung an VASP, das ausschließlich GP5 Motive beinhaltet. Die C-terminal verlängerte SH3 Domäne, die in der alternativ gespleißten αII-Spektrin Variante SH3i vorkommt, bindet stärker an Ena/VASP Proteine als die übliche αII-Spektrin Isoform und bereits die Expression der isolierten Domäne alleine ist für eine Kolokalisation mit Ena/VASP Proteinen in lebenden Endothelzellen ausreichend. Letztlich konnten wir zeigen, dass die Tetramerisierung der Ena/VASP Proteine unentbehrlich für die Interaktion mit allen untersuchten αII-Spektrin Isoformen ist.
urn:nbn:de:bvb:20-opus-154481
X 127333
CC BY-NC-ND: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell, Keine Bearbeitungen 4.0 International
Kristin Offner
mul
uncontrolled
SH3
eng
uncontrolled
proteininteraction
mul
uncontrolled
Mena
mul
uncontrolled
VASP
deu
uncontrolled
Proteininteraktion
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15448/offner_kristin_SH3.pdf
14312
2015
eng
7235
6
article
1
2017-01-23
--
--
Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue
Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing \(\beta\)\(_{1}\)-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities.
Nature Communications
10.1038/ncomms8235
urn:nbn:de:bvb:20-opus-143127
Nature Communications 6:7235 (2015). DOI: 10.1038/ncomms8235
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Linda S. Hoffmann
Jennifer Etzrodt
Lena Willkomm
Abhishek Sanyal
Ludger Scheja
Alexander W. C. Fischer
Johannes-Peter Stasch
Wilhelm Bloch
Andreas Friebe
Joerg Heeren
Alexander Pfeifer
eng
uncontrolled
decompensated heart failure
eng
uncontrolled
mitochondrial biogenesis
eng
uncontrolled
pulmonary hypertension
eng
uncontrolled
nitric oxide
eng
uncontrolled
erectile dysfunction
eng
uncontrolled
beige adipocytes
eng
uncontrolled
fat development
eng
uncontrolled
cGMP
eng
uncontrolled
riociguat
eng
uncontrolled
white
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14312/009_Hoffmann_NATURE_COMM.pdf
14796
2016
eng
e0167033
11
11
article
1
2017-05-05
--
--
PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling
Aim
Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.
Materials and Methods
In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes.
Results
In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.
Conclusions
PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.
PLoS One
10.1371/journal.pone.0167033
urn:nbn:de:bvb:20-opus-147967
PLoS ONE 11(11): e0167033. https://doi.org/10.1371/journal.pone.0167033
Hariharan Subramanian
Frank Döring
Sina Kollert
Natalia Rukoyatkina
Julia Sturm
Stepan Gambaryan
Angelika Stellzig-Eisenhauer
Philipp Meyer-Marcotty
Martin Eigenthaler
Erhard Wischmeyer
eng
uncontrolled
phosphorylation
eng
uncontrolled
xenopus oocytes
eng
uncontrolled
calcium signaling
eng
uncontrolled
intracellular receptors
eng
uncontrolled
mutation
eng
uncontrolled
teeth
eng
uncontrolled
tooth eruption
eng
uncontrolled
transfection
Chirurgie und verwandte medizinische Fachrichtungen
open_access
Institut für Klinische Biochemie und Pathobiochemie
Physiologisches Institut
Poliklinik für Kieferorthopädie
Förderzeitraum 2016
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14796/Subramanian_journal.pone.0167033.pdf
14660
2016
eng
140
13
article
1
2017-03-31
--
--
Combined [\(^{18}\)F]DPA-714 micro-positron emission tomography and autoradiography imaging of microglia activation after closed head injury in mice
Background
Traumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry.
Methods
A weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo μPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [\(^{18}\)F]DPA-714 was performed on day 1, 7, and 16 and [\(^{18}\)F]FDG-μPET imaging for energy metabolism on days 2–5 after trauma using freshly synthesized radiotracers. Immediately after [\(^{18}\)F]DPA-714-μPET imaging on days 7 and 16, cellular identity of the [\(^{18}\)F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1.
Results
Functional outcome correlated with focal brain lesions, gliosis, and axonal injury. [\(^{18}\)F]DPA-714-μPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [\(^{18}\)F]FDG uptake on days 2–5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [\(^{18}\)F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [\(^{18}\)F]DPA-714 was not increased in autoradiography or in μPET imaging.
Conclusions
[\(^{18}\)F]DPA-714 uptake in μPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI.
Journal of Neuroinflammation
10.1186/s12974-016-0604-9
urn:nbn:de:bvb:20-opus-146606
Journal of Neuroinflammation (2016) 13:140 DOI 10.1186/s12974-016-0604-9
680966
Ina Israel
Andrea Ohsiek
Ehab Al-Momani
Christiane Albert-Weissenberger
Christian Stetter
Stine Mencl
Andreas K. Buck
Christoph Kleinschnitz
Samuel Samnick
Anna-Leena Sirén
eng
uncontrolled
neuroinflammation
eng
uncontrolled
TBI
eng
uncontrolled
immunohistochemistry
eng
uncontrolled
weight drop
eng
uncontrolled
PET
eng
uncontrolled
diffuse
eng
uncontrolled
focal
eng
uncontrolled
TSPO
eng
uncontrolled
autoradiography
eng
uncontrolled
IBA-1
Medizin und Gesundheit
open_access
Neurochirurgische Klinik und Poliklinik
Klinik und Poliklinik für Nuklearmedizin
Physiologisches Institut
Neurologische Klinik und Poliklinik
OpenAIRE
Förderzeitraum 2016
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14660/Israel_10.1186_s12974-016-0604-9.pdf
14622
2016
eng
doctoralthesis
1
2017-03-28
--
0017-03-28
Functional and genetic dissection of mechanosensory organs of \(Drosophila\) \(melanogaster\)
Funktionelle und genetische Analyse von mmechanosensorischen Organe in \(Drosophila\) \(melanogaster\)
In Drosophila larvae and adults, chordotonal organs (chos) are highly versatile mechanosensors
that are essential for proprioception, touch sensation and hearing. Chos share molecular,
anatomical and functional properties with the inner ear hair cells of mammals. These multiple
similarities make chos powerful models for the molecular study of mechanosensation.
In the present study, I have developed a preparation to directly record from the sensory neurons
of larval chos (from the lateral chos or lch5) and managed to correlate defined mechanical inputs
with the corresponding electrical outputs. The findings of this setup are described in several case
studies.
(1) The basal functional lch5 parameters, including the time course of response during continuous
mechanical stimulation and the recovery time between successive bouts of stimulation, was
characterized.
(2) The calcium-independent receptor of α-latrotoxin (dCIRL/Latrophilin), an Adhesion class G
protein-coupled receptor (aGPCR), is identified as a modulator of the mechanical signals
perceived by lch5 neurons. The results indicate that dCIRL/Latrophilin is required for the
perception of external and internal mechanical stimuli and shapes the sensitivity of neuronal
mechanosensation.
(3) By combining this setup with optogenetics, I have confirmed that dCIRL modulates lch5
neuronal activity at the level of their receptor current (sensory encoding) rather than their ability
to generate action potentials.
(4) dCIRL´s structural properties (e.g. ectodomain length) are essential for the mechanosensitive
properties of chordotonal neurons.
(5) The versatility of chos also provides an opportunity to study multimodalities at multiple levels.
In this context, I performed an experiment to directly record neuronal activities at different
temperatures. The results show that both spontaneous and mechanically evoked activity increase
in proportion to temperature, suggesting that dCIRL is not required for thermosensation in chos.
These findings, from the development of an assay of sound/vibration sensation, to neuronal
signal processing, to molecular aspects of mechanosensory transduction, have provided the first
insights into the mechanosensitivity of dCIRL.
In addition to the functional screening of peripheral sensory neurons, another
electrophysiological approach was applied in the central nervous system: dCIRL may impact the
excitability of the motor neurons in the ventral nerve cord (VNC). In the second part of my work,
whole-cell patch clamp recordings of motor neuron somata demonstrated that action potential
firing in the dCirl\(^K\)\(^O\) did not differ from control samples, indicating comparable membrane
excitability.
In Drosophila Larven, sowie in adulten Tieren, sind die Chordotonalorgane (Chos) sehr vielseitige
Mechanosensoren und von wesentlicher Bedeutung für die Propriozeption, das Tastgefühl und die auditive Wahrnehmung. Chos teilen molekulare, anatomische und funktionelle Eigenschaften mit Innenohrhaarzellen der Säugetiere und machen sie somit zu leistungsstarken Modellen um molekulare Mechanismen der Mechanosensorik zu untersuchen. In der vorliegenden Studie habe ich ein Präparat entwickelt, um direkt von sensorischen Neuronen der larvalen Chos (von lateralen Chos oder lch5) abzuleiten und definierte mechanische Eingänge mit den korrelierenden elektrischen Ausgängen zu verbinden. Im Folgenden sind die Ergebnisse dieses experimentellen Setups zusammengefasst.
(1) Die basalen funktionellen Parameter von lch5 insbesondere der Zeitverlauf der Reaktion während kontinuierlicher mechanischer Stimulation und die Erholungszeit zwischen aufeinanderfolgenden Stimulationen wurden bestimmt.
(2) Der Calcium-unabhängige Rezeptor von α-Latrotoxin (dCIRL/Latrophilin), ein Adhäsion Klasse G-Protein-gekoppelter Rezeptor (GPCR) wurde als Modulator der von Ich5 Neuronen perzipierten mechanischen Signale identifiziert. Die Ergebnisse zeigen, dass dCIRL/Latrophilin für die Wahrnehmung der externen und internen mechanischen Reize erforderlich ist und die Empfindlichkeit neuronaler Mechanosensorik modelliert.
(3) Mit Hilfe optogenetischer Werkzeuge konnte ich bestätigen, dass dCIRL die Aktivität von lch5 Neuronen auf Ebene des Rezeptorstroms (sensorische Kodierung) und nicht der Generierung von Aktionspotentialen moduliert.
(4) Die strukturellen Eigenschaften von dCIRL (z.B. Ektodomänenlänge) sind wesentlich für die mechanosensitiven Eigenschaften von Chos.
(5) Die Vielseitigkeit der Chos bietet des Weiteren die Möglichkeit, Multimodalitäten auf mehreren Ebenen zu untersuchen. In diesem Zusammenhang wurde die neuronale Aktivität der Chos bei verschiedenen Temperaturen analysiert. Die Ergebnisse zeigen, dass sich sowohl spontane als auch mechanisch evozierte Aktivität im Verhältnis zur Temperatur erhöhen, was
darauf hindeutet, dass dCIRL keine Rolle in der Temperaturwahrnehmung spielt.
Diese Erkenntnisse, von der Entwicklung des Präparats der Ton/Vibrations Wahrnehmung, über
die neuronalen Signalverarbeitung bis hin zu molekularen Aspekten der Mechanotransduktion, haben erste Einblicke in die Mechanosensitivität von dCIRL gewährt.
Neben der funktionellen Charakterisierung peripherer sensorischer Neurone wurde ein weiterer elektrophysiologischer Ansatz im larvalen Zentralnervensystem gewählt, um zu untersuchen, ob sich dCIRL auf die Erregbarkeit motorischer Nervenzellen im Strickleiternervensystem (VNC) auswirkt. Im zweiten Teil meiner Arbeit wird mit Hilfe des whole-cell-patch-clamp-Verfahrens gezeigt, dass die Aktionspotentialfrequenz in Motoneuronen von dCirl\(^K\)\(^O\) Mutanten ähnlich derer
von Kontrolltieren ist, d.h. ihre Membranerregbarkeit ist vergleichbar.
urn:nbn:de:bvb:20-opus-146220
X 127100
Chonglin Guan
deu
swd
Taufliege
eng
uncontrolled
Drosophila
deu
uncontrolled
Mechanosensation
deu
uncontrolled
Adhesion-GPCR
eng
uncontrolled
Electrophysiology
deu
swd
Mechanorezeptor
deu
swd
Elektrophysiologie
eng
uncontrolled
Chordontonal organ
Biowissenschaften; Biologie
Medizin und Gesundheit
open_access
Graduate School of Life Sciences
Physiologisches Institut
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14622/Guan_Chonglin_Drosophila.pdf
14327
2016
eng
doctoralthesis
1
2017-01-24
--
2017-01-20
Effects of Transgenic Expression of Botulinum Toxins in Drosophila
Effekte der transgenen Expression von Botulinumtoxinen in Drosophila
Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.
Die verschiedenen Toxine der Bakterienspezies Clostridium (Botulinumtoxine und Tetanustoxin) interferieren mit Neuroexozytose durch Proteolyse der SNARE-Proteine. Wir haben transgene Fliegen generiert, die die Möglichkeit bieten konditional verschiedene Botulinumtoxine zu exprimieren. Durch biochemische Untersuchungen und Verhaltensexperimente haben wir das Potential dieser Toxine als Werkzeuge für die Analyse von Synapsen- und Netzwerkfunktion in Drosophila evaluiert. Durch Western Blot-Analysen stellten wir eine variierende Proteolysierbarkeit der Drosophila SNARE-Substrate durch die verschiedenen Botulinumtoxine dar. In Vivo untersuchten wir die Auswirkungen einer Zell-spezifischen Expression auf die Motorik in Larven und auf die Sehfähigkeit in adulten Fliegen. Unsere Resultate zeigen, dass Botulinumtoxin Typ B und C vielversprechende Alternativen zu etablierten molekularen Werkzeugen, wie Tetanustoxin, darstellen, um synaptische Transmission oder höhere Netzwerkfunktionen aufzuschlüsseln. Hierbei führt Botulinumtoxin Typ B zu einem spezifischen Verlust von neuronaler Aktivität, während Botulinumtoxin Typ C mit nicht Neuronen-spezifischer Zellfunktion interferiert.
urn:nbn:de:bvb:20-opus-143279
X 127043
Philipp Backhaus
deu
swd
Botulinustoxin
deu
swd
Drosophila
deu
swd
Synaptische Transmission
eng
uncontrolled
Behavioral neuroscience
eng
uncontrolled
molecular neuroscience
eng
uncontrolled
neurotoxins
eng
uncontrolled
SNARE proteins
eng
uncontrolled
neurotransmission
Einzelne physiologische Systeme bei Tieren
Humanphysiologie
open_access
Physiologisches Institut
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14327/Backhaus_Philipp_Drosophila.pdf
14193
2015
eng
e1257
3
article
1
2016-12-16
--
--
Temporal and spatial strategies in an active place avoidance task on Carousel: a study of effects of stability of arena rotation speed in rats
The active place avoidance task is a dry-arena task used to assess spatial navigation and memory in rodents. In this task, a subject is put on a rotating circular arena and avoids an invisible sector that is stable in relation to the room. Rotation of the arena means that the subject's avoidancemust be active, otherwise the subject will be moved in the to-be-avoided sector by the rotation of the arena and a slight electric shock will be administered. The present experiment explored the effect of variable arena rotation speed on the ability to avoid the to-be-avoided sector. Subjects in a group with variable arena rotation speed learned to avoid the sector with the same speed and attained the same avoidance ability as rats in a group with a stable arena rotation speed. Only a slight difference in preferred position within the room was found between the two groups. No difference was found between the two groups in the dark phase, where subjects could not use orientation cues in the room. Only one rat was able to learn the avoidance of the to-be-avoided sector in this phase. The results of the experiment suggest that idiothetic orientation and interval timing are not crucial for learning avoidance of the to-be-avoided sector. However, idiothetic orientation might be sufficient for avoiding the sector in the dark.
PeerJ
10.7717/peerj.1257
urn:nbn:de:bvb:20-opus-141931
PeerJ 3:e1257; DOI 10.7717/peerj.1257
Štěpán Bahník
Aleš Stuchlík
eng
uncontrolled
navigation
eng
uncontrolled
interval timing
eng
uncontrolled
rats
eng
uncontrolled
morris water maze
eng
uncontrolled
hippocampal-neurons
eng
uncontrolled
D2 receptors
eng
uncontrolled
animal model
eng
uncontrolled
acute MK-801
eng
uncontrolled
memory
eng
uncontrolled
behavior
eng
uncontrolled
dissociation
eng
uncontrolled
flexibility
eng
uncontrolled
spatial navigation
eng
uncontrolled
substratal idiothetic navigation
eng
uncontrolled
inertial idiothetic navigation
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14193/Bahnik.pdf
10735
2014
eng
doctoralthesis
1
2014-12-17
--
2014-07-30
Identification of SPRED2 as a Novel Regulator of Hypothalamic-Pituitary-Adrenal Axis Activity and of Body Homeostasis
SPRED2 - Ein neuer Regulator der Hypothalamus-Hypophysen-Nebennierenrindenachse und der Hormonbalance
SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype.
The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1.
Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage.
Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis.
To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.
SPRED-Proteine sind Inhibitoren des hochkonservierten und in allen Geweben verbreiteten Ras/ERK/MAPK-Signalwegs, welcher Proliferation, Differenzierung und das Wachstum von Zellen reguliert. Um physiologische Konsequenzen der SPRED2-Defizienz im lebenden Modellorganismus aufzuklären, haben wir SPRED2-KO-Mäuse mithilfe der „gene trap“-Methode generiert. Eine erste Studie zur phänotypischen Charakterisierung mit KO-Mäusen bis zu einem Alter von fünf Monaten identifizierte SPRED2 als Regulator der Chondrozytendifferenzierung und des Knochenwachstums. So bewirkt der Verlust der SPRED2-Proteinfunktion eine erhöhte FGFR-vermittelte ERK-Aktivität, was wiederum einen Hypochondroplasie-ähnlichen Minderwuchs verursacht. Allerdings offenbarten Langzeitbeobachtungen älterer KO-Mäuse einen im Allgemeinen sehr schlechten Gesundheitszustand und weitere facettenreiche Symptome, darunter exzessives Putzverhalten mit schweren, selbst zugefügten Wunden, einen abnorm hohen täglichen Wasserkonsum, klare morphologische Anzeichen einer Nierenschädigung und eine reduzierte Überlebenswahrscheinlichkeit durch plötzlichen Tod. Ziel dieser Studie war es, basierend auf unseren Beobachtungen, einen Auslöser für diesen komplexen und vielseitigen Phänotyp zu finden.
Die beobachtete Nierendegeneration in unseren SPRED2-KO-Mäusen war auf eine Hydronephrose zurückzuführen, welche durch schwere Atrophie des Nierengewebes und Apoptose von Nierentubuluszellen gekennzeichnet war. Aufgrund des Nierenschadens haben wir Trinkverhalten und gängige Serumparameter analysiert. Trotz der Polydipsie, die sich durch eine nahezu verdoppelte tägliche Wasseraufnahme manifestierte, konnten signifikant erhöhte Na+- und Cl--Werte und die daraus resultierende Hyperosmolalität im Serum der SPRED2-KOs nicht kompensiert werden. Weil Salz- und Wasserhaushalt zum größten Teil unter der hormonellen Kontrolle von Aldosteron und ADH stehen, haben wir beide Hormonspiegel untersucht. Während die ADH-Werte im Serum von WT- und KO-Mäusen vergleichbar waren, insbesondere nach experimentellem Wasserentzug und einem extremen Verlust von Körperflüssigkeit, waren die Serumspiegel von Aldosteron in den SPRED2-KO-Mäusen verdoppelt. Die systematische Untersuchung übergeordneter regulatorischer Hormonachsen ergab, dass sich der Hyperaldosteronismus unabhängig von einer erhöhten Aktivität des Renin-Angiotensin-Systems entwickelte, da die Serum-Ang II-Spiegel in den SPRED2-KOs etwa um die Hälfte reduziert waren. Die Expression der Aldosteronsynthase in der Nebenniere war jedoch wesentlich erhöht. Für Kortikosteron, das wie Aldosteron von der Nebennierenrinde freigesetzt wird, konnten wir ebenfalls mehr als doppelt so hohe Werte im Serum der KO-Tiere detektieren. Die Aldosteron-Produktion steht, ähnlich wie bei Kortikosteron, zumindest teilweise unter der Kontrolle des hypophysären Hormons ACTH, dessen Sekretion wiederum übergeordnet durch die Freisetzung von CRH aus dem Hypothalamus geregelt wird. Tatsächlich war die Stresshormon-Sekretion entlang dieser gesamten Hypothalamus-Hypophysen-Nebennierenrinden-Achse erhöht, da Serum-ACTH, das mittlere, hypophysäre Hormon, und hypothalamisches CRH, der übergeordnete hormonelle Induktor der HPA-Achse, in den SPRED2-KOs auch um 30% erhöht waren. Zusätzlich waren die ERK-Aktivität ebenso wie die CRH-mRNA-Spiegel im paraventrikulären Nukleus des Hypothalamus in unseren SPRED2-KO-Mäusen deutlich höher. In vitro Studien mit der Hypothalamus-Zelllinie mHypoE-44 zeigten weiterhin, dass sowohl SPRED1 als auch SPRED2 die Aktivität des CRH-Promotors, die CRH-Sekretion und die Ets-Faktor-abhängige CRH-Transkription reduzieren können. Passend dazu enthält die CRH-Promotorregion zahlreiche verschiedene Bindungsstellen für Transkriptionsfaktoren der Ets-Familie, speziell für Ets1.
Somit zeigt diese Studie zum ersten Mal, dass die durch SPRED2-vermittelte Hemmung der Ras/ERK/MAPK-Signalkaskade mittels Unterdrückung der ERK-Aktivität zu einer Herunterregulation der Ets1-Faktor-abhängigen Transkription führt, was eine Hemmung der CRH-Promotoraktivität, der CRH-Transkription und der CRH-Freisetzung aus dem Hypothalamus zur Folge hat. Die daraus resultierende Hyperaktivität der gesamten HPA-Achse in unseren SPRED2-KO-Mäusen spiegelt eine erhöhte endogene Stress-Reaktion wider und äußert sich durch übermäßiges Putzverhalten und durch selbst zugefügte Hautläsionen auf der einen Seite; auf der anderen Seite erklärt dies, in Kombination mit der erhöhten Aldosteronsynthase-Expression, den Hyperaldosteronismus und das damit verbundene Ungleichgewicht in Salz- und Wasserhaushalt. Weiterhin tragen sowohl Hyperaldosteronismus als auch Polydipsie sehr wahrscheinlich zu den beobachteten Nierenschädigungen bei.
Zusammengefasst ist diese Studie ein erster Hinweis, dass SPRED2 wesentlich an der adäquaten Regulation der HPA-Achsen-Aktivität beteiligt ist und essentiell ist für die Aufrechterhaltung der Homöostase im Körper.
Um die Folgen von SPRED2-Defizienz in Mäusen und vor allem im Menschen weiter aufzuklären und zu vergleichen, erforschen wir in zwei Folgeprojekten die Funktion von SPRED2 speziell im Gehirn und im Herzen und führen parallel ein genetisches Screening zur Identifikation von funktionellen SPRED2-Mutationen im Menschen durch.
urn:nbn:de:bvb:20-opus-107355
X 126685
Melanie Ullrich
deu
swd
Renin-Angiotensin-System
deu
swd
Spred-Proteine
deu
swd
MAP-Kinase
deu
swd
Hypophysen-Zwischenhirn-System
deu
swd
Knockout <Molekulargenetik>
eng
uncontrolled
SPRED2
eng
uncontrolled
ERK
eng
uncontrolled
MAP Kinase Signaling
eng
uncontrolled
HPA Axis
eng
uncontrolled
Renin Angiotensin System
eng
uncontrolled
Knockout mouse
eng
uncontrolled
Spred Protein
deu
uncontrolled
Hypothalamisch-hypophysäre Achse
deu
uncontrolled
Renin-Angiotensin-Aldosteron-System
deu
uncontrolled
MAP-Kinase
deu
uncontrolled
Gen-Knockout
Biowissenschaften; Biologie
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/10735/Dissertation_Ullrich_Melanie.pdf
13058
2013
eng
16
Article ID 265182
article
1
2016-03-23
--
--
Identification of PDZ domain containing proteins interacting with \(Ca_v1.2\) and PMCA4b
PDZ (PSD-95/Disc large/Zonula occludens-1) protein interaction domains bind to cytoplasmic protein C-termini of transmembrane proteins. In order to identify new interaction partners of the voltage-gated L-type \(Ca^{2+}\) channel Cav1.2 and the plasma membrane \(Ca^{2+}\) ATPase 4b (PMCA4b), we used PDZ domain arrays probing for 124 PDZ domains. We confirmed this byGST pulldowns and immunoprecipitations. In PDZ arrays, strongest interactionswith \(Ca_v1.2\) and PMCA4b were found for the PDZ domains of SAP-102, MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. We observed binding of the \(Ca_v1.2\) C-terminus to PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2 and its known interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of \(Ca_v1.2\) and PMCA4b with NHERF1/2, CASK,MAST-205 and MAGI-3 viaimmunoprecipitation. We also verified the interaction of \(Ca_v1.2\) and nNOS and hypothesized that nNOS overexpression might reduce \(Ca^{2+}\) influx through \(Ca_v1.2\). To address this, we measured \(Ca^{2+}\) currents in HEK 293 cells co-expressing \(Ca_v1.2\) and nNOS and observed reduced voltage-dependent \(Ca_v1.2\) activation. Taken together, we conclude that \(Ca_v1.2\) and PMCA4b bind promiscuously to various PDZ domains, and that our data provides the basis for further investigation of the physiological consequences of these interactions.
ISRN Cell Biology
10.1155/2013/265182
urn:nbn:de:bvb:20-opus-130585
ISRN Cell Biology Volume 2013, Article ID 265182, 16 pages http://dx.doi.org/10.1155/2013/265182
Doreen Korb
Priscilla Y. Tng
Vladimir M. Milenkovic
Nadine Reichhart
Olaf Strauss
Oliver Ritter
Tobias Fischer
Peter M. Benz
Kai Schuh
eng
uncontrolled
Cell
Humanphysiologie
open_access
Physiologisches Institut
Medizinische Klinik und Poliklinik I
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13058/052_Korb_Tng_Milenkovic.pdf
13469
2013
eng
1233–1244
34
article
1
2016-06-11
--
--
Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I
Aims: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism.
Methods and results: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the \([Ca^{2+}]_i\)-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte \(Ca^{2+}_i\) homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, \(Ca^{2+}_i\)-handling, and contractility via cGKI.
Conclusion: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte \(Ca^{2+}_i\) handling and contractility.
European Heart Journal
urn:nbn:de:bvb:20-opus-134693
European Heart Journal (2013) 34, 1233–1244 doi:10.1093/eurheartj/ehr445
Stefan Frantz
Michael Klaiber
Hideo A. Baba
Heinz Oberwinkler
Katharina Völker
Birgit Gaßner
Barbara Bayer
Marco Abeßer
Kai Schuh
Robert Feil
Franz Hofmann
Michaela Kuhn
eng
uncontrolled
cyclic
eng
uncontrolled
GMPcGMP-dependent protein kinase I
eng
uncontrolled
cardiac hypertrophy
eng
uncontrolled
natriuretic peptide
eng
uncontrolled
Ca2+i handling
Medizin und Gesundheit
open_access
Physiologisches Institut
Medizinische Klinik und Poliklinik I
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13469/146_Frantz_European_Heart.pdf
12880
2013
eng
1407-1413
5
3
article
1
2016-03-07
--
--
Hebbian Plasticity Guides Maturation of Glutamate Receptor Fields In Vivo
Synaptic plasticity shapes the development of functional neural circuits and provides a basis for cellular models of learning and memory. Hebbian plasticity describes an activity-dependent change in synaptic strength that is input-specific and depends on correlated pre- and postsynaptic activity. Although it is recognized that synaptic activity and synapse development are intimately linked, our mechanistic understanding of the coupling is far from complete. Using Channelrhodopsin-2 to evoke activity in vivo, we investigated synaptic plasticity at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation increased postsynaptic sensitivity by promoting synapse-
specific recruitment of GluR-IIA-type glutamate receptor subunits into postsynaptic receptor fields. Conversely, GluR-IIA was rapidly removed from synapses whose activity failed to evoke substantial postsynaptic depolarization. Uniting these results with developmental GluR-IIA dynamics provides a comprehensive physiological concept of how Hebbian plasticity guides synaptic maturation and sparse transmitter release controls the stabilization of the molecular composition of individual synapses.
Cell Reports
10.1016/j.celrep.2013.04.003
urn:nbn:de:bvb:20-opus-128804
Cell Reports 3, 1407–1413. doi:10.1016/j.celrep.2013.04.003
Dmitrij Ljaschenko
Nadine Ehmann
Robert J. Kittel
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12880/020_Hebbian Plasticity Guides Maturation.pdf
12769
1993
eng
C31-34
Special Conference Issue
38
article
1
2016-02-23
--
--
Small reduction of capsaicin-induced neurogenic inflammation in human forearm skin by the glucocorticoid prednicarbate
No abstract available.
Agents Actions
urn:nbn:de:bvb:20-opus-127698
Agents Actions 38, Special Conference Issue (1993), C31-34
Deutsches Urheberrecht
R. Tafler
M. K. Herbert
R. F. Schmidt
K. H. Weis
Medizin und Gesundheit
open_access
Klinik für Anaesthesiologie (bis 2003)
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12769/X_Herbert_neurogenic-inflammation.pdf