The actin binding protein drebrin helps to protect against the development of seizure-like events in the entorhinal cortex.
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ABSTRACT:
SUBMITTER: Klemz A
PROVIDER: S-EPMC8060314 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 [1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 A from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 A). These results suggest that the "core" DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins.
| S-EPMC2866048 | biostudies-literature
Project description:Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton.Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer.Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.
| S-EPMC4850108 | biostudies-other
Project description:BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.
| S-EPMC3251523 | biostudies-literature
Project description:Single-unit recordings performed in temporal lobe epilepsy patients and in models of temporal lobe seizures have shown that interneurons are active at focal seizure onset. We performed simultaneous patch-clamp and field potential recordings in entorhinal cortex slices of GAD65 and GAD67 C57BL/6J male mice that express green fluorescent protein in GABAergic neurons to analyze the activity of specific interneuron (IN) subpopulations during acute seizure-like events (SLEs) induced by 4-aminopyridine (4-AP; 100 μm). IN subtypes were identified as parvalbuminergic (INPV, n = 17), cholecystokinergic (INCCK), n = 13], and somatostatinergic (INSOM, n = 15), according to neurophysiological features and single-cell digital PCR. INPV and INCCK discharged at the start of 4-AP-induced SLEs characterized by either low-voltage fast or hyper-synchronous onset pattern. In both SLE onset types, INSOM fired earliest before SLEs, followed by INPV and INCCK discharges. Pyramidal neurons became active with variable delays after SLE onset. Depolarizing block was observed in ∼50% of cells in each INs subgroup, and it was longer in IN (∼4 s) than in pyramidal neurons (<1 s). As SLE evolved, all IN subtypes generated action potential bursts synchronous with the field potential events leading to SLE termination. High-frequency firing throughout the SLE occurred in one-third of INPV and INSOM We conclude that entorhinal cortex INs are very active at the onset and during the progression of SLEs induced by 4-AP. These results support earlier in vivo and in vivo evidence and suggest that INs have a preferential role in focal seizure initiation and development.SIGNIFICANCE STATEMENT Focal seizures are believed to result from enhanced excitation. Nevertheless, we and others demonstrated that cortical GABAergic networks may initiate focal seizures. Here, we analyzed for the first time the role of different IN subtypes in seizures generated by 4-aminopyridine in the mouse entorhinal cortex slices. We found that in this in vitro focal seizure model, all IN types contribute to seizure initiation and that INs precede firing of principal cells. This evidence is in agreement with the active role of GABAergic networks in seizure generation.
| S-EPMC10027059 | biostudies-literature
Project description:Persistent transcriptional and morphological events in the nucleus accumbens (NAc) and other brain reward regions contribute to the long-lasting behavioral adaptations that characterize drug addiction. Opiate exposure reduces the density of dendritic spines on medium spiny neurons of the NAc; however, the underlying transcriptional and cellular events mediating this remain unknown. We show that heroin self-administration negatively regulates the actin-binding protein drebrin in the NAc. Using virus-mediated gene transfer, we show that drebrin overexpression in the NAc is sufficient to decrease drug seeking and increase dendritic spine density, whereas drebrin knockdown potentiates these effects. We demonstrate that drebrin is transcriptionally repressed by the histone modifier HDAC2, which is relieved by pharmacological inhibition of histone deacetylases. Importantly, we demonstrate that heroin-induced adaptations occur only in the D1+ subset of medium spiny neurons. These findings establish an essential role for drebrin, and upstream transcriptional regulator HDAC2, in opiate-induced plasticity in the NAc.
| S-EPMC6742638 | biostudies-literature
Project description:Fibrosis is mainly triggered by inflammation in various tissues, such as heart and liver tissues, and eventually leads to their subsequent dysfunction. Fibrosis is characterized by the excessive accumulation of extracellular matrix proteins (e.g., collagens) produced by myofibroblasts. The well-developed actin cytoskeleton of myofibroblasts, one of the main features differentiating them from resident fibroblasts in tissues under inflammatory conditions, contributes to maintaining their ability to produce excessive extracellular matrix proteins. However, the molecular mechanisms via which the actin cytoskeleton promotes the production of fibrosis-related genes in myofibroblasts remain unclear. In this study, we found, via single-cell analysis, that developmentally regulated brain protein (drebrin), an actin-binding protein, was specifically expressed in cardiac myofibroblasts with a well-developed actin cytoskeleton in fibrotic hearts. Moreover, our immunocytochemistry analysis revealed that drebrin promoted actin cytoskeleton formation and myocardin-related transcription factor-serum response factor signaling. Comprehensive single-cell analysis and RNA-Seq revealed that the expression of collagen triple helix repeat containing 1 (Cthrc1), a fibrosis-promoting secreted protein, was regulated by drebrin in cardiac myofibroblasts via myocardin-related transcription factor-serum response factor signaling. Furthermore, we observed the profibrotic effects of drebrin exerted via actin cytoskeleton formation and the Cthrc1 expression regulation by drebrin in liver myofibroblasts (hepatic stellate cells). Importantly, RNA-Seq demonstrated that drebrin expression levels increased in human fibrotic heart and liver tissues. In summary, our results indicated that the well-developed actin cytoskeleton and Cthrc1 expression due to drebrin in myofibroblasts promoted cardiac and hepatic fibrosis, suggesting that drebrin is a therapeutic target molecule for fibrosis.
| S-EPMC9988570 | biostudies-literature
Project description:We investigated whether an anti-epileptic effect is obtained by selectively activating excitatory neurons expressing ChR2 under the promoter for the synaptic vesicular glutamate transporter 2 (VGLUT2). VGLUT2-expressing cells were optically stimulated while local field potential and whole-cell patch-clamp recordings were performed in mouse entorhinal cortical slices perfused with the proconvulsive compound 4-aminopyridine (4-AP). In control conditions, blue light flashes directly depolarized the majority of putative glutamatergic cells, which in turn synaptically excited GABAergic interneurons. During bath perfusion with 4-AP, photostimuli triggered a fast EPSP-IPSP sequence which was often followed by tonic-clonic seizure-like activity closely resembling spontaneous ictal discharges. The GABAA-receptor antagonist gabazine blocked the progression of both light-induced and spontaneous seizures. Surprisingly, prolonged photostimuli delivered during ongoing seizures caused a robust interruption of synchronous discharges. Such break was correlated with a membrane potential depolarization block in principal cells, while putative GABAergic interneurons changed their firing activity from a burst-like to an irregular single-spike pattern. These data suggest that photostimulation of glutamatergic neurons triggers seizure-like activity only in the presence of an intact GABAergic transmission and that selectively activating the same glutamatergic cells robustly interrupts ongoing seizures by inducing a strong depolarization block, resulting in the disruption of paroxysmal burst-like firing.
| S-EPMC5322365 | biostudies-literature
Project description:Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.
| S-EPMC3733845 | biostudies-literature
Project description:The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.
| S-EPMC5309812 | biostudies-literature
Project description:BACKGROUND:Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury that has a high mortality rate and leads to substantial healthcare costs. MicroRNA-124-3p (miR-124-3p) helps to suppress inflammation during a pulmonary injury. However, its mechanism of action is largely unknown, and its role in ARDS remains to be determined. METHODS:Mice and NR8383 cells were exposed to lipopolysaccharides (LPS) to induce ARDS, and their miR-124-3p levels were determined. After a miRNA agomir was administrated to the mice, their pulmonary injuries were evaluated by H&E staining and assays for peripheral inflammatory cytokine levels. The direct interaction between miR-124-3p and p65 was predicted, and then confirmed by a luciferase activity assay. The role played by miRNA-124-3p in regulating p65 expression was further examined by transfection with its agomir, and its role in cell apoptosis was investigated by observing the effects of miRNA overexpression in vitro and in vivo. RESULTS:After exposure to LPS, there was a consistent decrease in miR-124-3p expression in the lungs of mice and in NR8383 cells. After treatment with the miR-124-3p agomir, the degrees of pulmonary injury (e.g. alveolar hemorrhage and interstitial edema), and the increases in IL-1?, IL-6, and TNF-? levels induced by LPS were significantly attenuated. Overexpression of miR-124-3p in NC8383 cells and lung tissues significantly suppressed LPS-induced p65 expression and cell apoptosis. CONCLUSIONS:These results suggest that miR-124-3p directly targeted p65, and thereby decreased the levels of inflammation and pulmonary injury in a mouse model of ARDS.
| S-EPMC7253404 | biostudies-literature