Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aβ ligation to β1-integrin conformers.
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ABSTRACT:
SUBMITTER: Woo JA
PROVIDER: S-EPMC4423194 | biostudies-other | 2015 Jun
REPOSITORIES: biostudies-other
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Project description:The accumulation of amyloid-β protein (Aβ) is an early event associated with synaptic and mitochondrial damage in Alzheimer's disease (AD). Recent studies have implicated the filamentous actin (F-actin) severing protein, Cofilin, in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of the AD pathogenic process and how Aβ impinges its signals to Cofilin from the neuronal surface are unknown. In this study, we found that Aβ42 oligomers (Aβ42O, amyloid-β protein 1-42 oligomers) bind with high affinity to low or intermediate activation conformers of β1-integrin, resulting in the loss of surface β1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of β1-integrin prevented Aβ42O-induced Cofilin activation, and allosteric modulation or activation of β1-integrin significantly reduced Aβ42O binding to neurons while blocking Aβ42O-induced reactive oxygen species (ROS) production, mitochondrial dysfunction, depletion of F-actin/focal Vinculin, and apoptosis. Cofilin, in turn, was required for Aβ42O-induced loss of cell surface β1-integrin, disruption of F-actin/focal Talin-Vinculin, and depletion of F-actin-associated postsynaptic proteins. SSH1 reduction, which mitigated Cofilin activation, prevented Aβ42O-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, AD mouse model (APP (amyloid precursor protein)/PS1) brains contained increased SSH1/Cofilin and decreased SSH1/14-3-3 complexes, indicative of SSH1-Cofilin activation via release of SSH1 from 14-3-3. Finally, genetic reduction in Cofilin rescued APP/Aβ-induced synaptic protein loss and gliosis in vivo as well as deficits in long-term potentiation (LTP) and contextual memory in APP/PS1 mice. These novel findings therefore implicate the essential involvement of the β1-integrin-SSH1-Cofilin pathway in mitochondrial and synaptic dysfunction in AD.
| S-EPMC4423195 | biostudies-literature
Project description:AimsAmyloid-β (Aβ) oligomers trigger synaptic degeneration that precedes plaque and tangle pathology. However, the signalling molecules that link Aβ oligomers to synaptic pathology remain unclear. Here, we addressed the potential role of RAPGEF2 as a novel signalling molecule in Aβ oligomer-induced synaptic and cognitive impairments in human-mutant amyloid precursor protein (APP) mouse models of Alzheimer's disease (AD).MethodsTo investigate the role of RAPGEF2 in Aβ oligomer-induced synaptic and cognitive impairments, we utilised a combination of approaches including biochemistry, molecular cell biology, light and electron microscopy, behavioural tests with primary neuron cultures, multiple AD mouse models and post-mortem human AD brain tissue.ResultsWe found significantly elevated RAPGEF2 levels in the post-mortem human AD hippocampus. RAPGEF2 levels also increased in the transgenic AD mouse models, generating high levels of Aβ oligomers before exhibiting synaptic and cognitive impairment. RAPGEF2 upregulation activated the downstream effectors Rap2 and JNK. In cultured hippocampal neurons, oligomeric Aβ treatment increased the fluorescence intensity of RAPGEF2 and reduced the number of dendritic spines and the intensities of synaptic marker proteins, while silencing RAPGEF2 expression blocked Aβ oligomer-induced synapse loss. Additionally, the in vivo knockdown of RAPGEF2 expression in the AD hippocampus prevented cognitive deficits and the loss of excitatory synapses.ConclusionsThese findings demonstrate that the upregulation of RAPGEF2 levels mediates Aβ oligomer-induced synaptic and cognitive disturbances in the AD hippocampus. We propose that an early intervention regarding RAPGEF2 expression may have beneficial effects on early synaptic pathology and memory loss in AD.
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Project description:Mitochondrial dysfunction and synaptic damage are important features of Alzheimer's disease (AD) associated with amyloid ? (A?) and tau. We reported previously that the scaffolding protein RanBP9, which is overall increased in brains of patients with AD and in mutant APP transgenic mice, simultaneously promotes A? generation and focal adhesion disruption by accelerating the endocytosis of APP and ?1-integrin, respectively. Moreover, RanBP9 induces neurodegeneration in vitro and in vivo and mediates A?-induced neurotoxicity. Here we show in primary hippocampal neurons that RanBP9 potentiates A?-induced reactive oxygen species (ROS) overproduction, apoptosis, and calcium deregulation. Analyses of calcium-handling measures demonstrate that RanBP9 selectively delays the clearance of cytosolic Ca(2+) mediated by the mitochondrial calcium uniporter through a process involving the translocation of cofilin into mitochondria and oxidative mechanisms. Further, RanBP9 retards the anterograde axonal transport of mitochondria in primary neurons and decreases synaptic mitochondrial activity in brain. These data indicate that RanBP9, cofilin, and A? mimic and potentiate each other to produce mitochondrial dysfunction, ROS overproduction, and calcium deregulation, which leads to neurodegenerative changes reminiscent of those seen in AD.
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Project description:Myt3 is a prosurvival factor in pancreatic islets; however, its role in islet-cell development is not known. Here, we demonstrate that myelin transcription factor 3 (Myt3) is expressed in migrating islet cells in the developing and neonatal pancreas and thus sought to determine whether Myt3 plays a role in this process. Using an ex vivo model of islet-cell migration, we demonstrate that Myt3 suppression significantly inhibits laminin-V/integrin-β1-dependent α- and β-cell migration onto 804G, and impaired 804G-induced F-actin and E-cadherin redistribution. Exposure of islets to proinflammatory cytokines, which suppress Myt3 expression, had a similar effect, whereas Myt3 overexpression partially rescued the migratory ability of the islet cells. We show that loss of islet-cell migration, due to Myt3 suppression or cytokine exposure, is independent of effects on islet-cell survival or proliferation. Myt3 suppression also had no effect on glucose-induced calcium influx, F-actin remodeling or insulin secretion by β-cells. RNA-sequencing (RNA-seq) analysis of transduced islets showed that Myt3 suppression results in the up-regulation of Tgfbi, a secreted diabetogenic factor thought to impair cellular adhesion. Exposure of islets to exogenous transforming growth factor β-induced (Tgfbi) impaired islet-cell migration similar to Myt3 suppression. Taken together, these data suggest a model by which cytokine-induced Myt3 suppression leads to Tgfbi de-repression and subsequently to impaired islet-cell migration, revealing a novel role for Myt3 in regulating islet-cell migration.
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Project description:Synaptic dysfunction and neuronal excitatory/inhibitory imbalance have been implicated in Alzheimer's disease (AD) pathogenesis. Although intensive studies have been focused on the excitatory synaptic system, much less is known concerning the mechanisms mediating inhibitory synaptic dysfunction in AD. We reported previously that protocadherin-?C5 (Pcdh-?C5), a member of clustered Pcdh-? subfamily of cadherin-type synaptic adhesion proteins, functions to promote GABAergic synaptic transmission. We reveal here that Pcdh-?C5 is enriched in vesicular GABA transporter-positive synaptic puncta and its expression levels are increased in neuronal hyperexcitation conditions, upon ?-amyloid (A?) treatment, and in amyloid precursor protein (APP)/presenilin-1 (PS1)-transgenic mice of both sexes. This is associated with elevated levels of GABAergic proteins and enhanced synaptic inhibition. Genetic knock-down experiments showed that Pcdh-?C5 modulates spontaneous synaptic currents and A?-induced synaptic alterations directly. Our results support a model in which Pcdh-?C5 senses neuronal hyperexcitation to augment GABAergic inhibition. This adaptive mechanism may be dysregulated under chronic excitation conditions such as AD, leading to aberrant Pcdh-?C5 expression and associated synaptic dysfunction.SIGNIFICANCE STATEMENT Synaptic dysfunction is causal for Alzheimer's disease (AD). Here, we reveal a novel pathway that contributes GABAergic synaptic dysfunction in AD mediated by protocadherin-?C5. Our study not only identifies a new mechanism mediating excitatory/inhibitory balance in AD, but may also offer a new target for potential therapeutic intervention.
| S-EPMC5607468 | biostudies-literature
Project description:Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca2+ signaling is believed to play an important role. However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix. Methods: Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca2+ signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca2+ signaling was assessed. Results: Ca2+ responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X-but not Piezo1-ion channels. The resultant Ca2+ influx caused dynamic changes in cell spread area. Conclusion: This approach to safely eliciting a Ca2+ response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.
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Project description:IntroductionThe overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The β1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival.MethodsWe developed a panel of HER2-overexpressing cell lines resistant to L, T, and the potent LT combination through long-term exposure and validated these models in 3D culture. Parental and L/T/LT-resistant cells were subject to HER2 and β1 integrin inhibitors in 3D and monitored for 12 days, followed by quantification of colony number. Parallel experiments were conducted where cells were either stained for Ki-67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or harvested for protein and analyzed by immunoblot. Results were subjected to statistical testing using analysis of variance and linear contrasts, followed by adjustment with the Sidak method.ResultsUsing multiple cell lines including BT474 and HCC1954, we reveal that in L and LT resistance, where phosphorylation of EGFR/HER1, HER2, and HER3 are strongly inhibited, kinases downstream of β1 integrin--including focal adhesion kinase (FAK) and Src--are up-regulated. Blockade of β1 by the antibody AIIB2 abrogates this up-regulation and functionally achieves significant growth inhibition of L and LT resistant cells in 3D, without dramatically affecting the parental cells. SiRNA against β1 as well as pharmacologic inhibition of FAK achieve the same growth inhibitory effect. In contrast, trastuzumab-resistant cells, which retain high levels of phosphorylated EGFR/HER1, HER2, and HER3, are only modestly growth-inhibited by AIIB2.ConclusionsOur data suggest that HER2 activity, which is suppressed in resistance involving L but not T alone, dictates whether β1 mediates an alternative pathway driving resistance. Our findings justify clinical studies investigating the inhibition of β1 or its downstream signaling moieties as strategies to overcome acquired L and LT resistance.
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