Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Formation of complex AChR aggregates in vitro requires α‐dystrobrevin

Efficient function at the neuromuscular junction requires high‐density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. α‐Dystrobrevin (αDB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of αDB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel‐like AChR aggregates on primary muscle cell cultures derived from αDB knockout (αDB‐KO) mice in the absence of nerve or agrin. In myotubes lacking αDB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of αDB are expressed in skeletal muscle: αDB1, αDB1(?), αDB2, αDB2(?), and αDB3. Expression of αDB1 or αDB1(?) in αDB‐KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of αDB2, αDB2(?), αDB3, or an αDB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that αDB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that αDB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in αDB‐KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Parathyroid hormone inhibits phosphorylation of mitogen‐activated protein kinase (MAPK) ERK1/2 through inhibition of c‐Raf and activation of MKP‐1 in osteoblastic cells

Parathyroid hormone (PTH) regulation of mitogen‐activated protein kinases (MAPK) ERK1/2 contributes to PTH regulation of osteoblast growth and apoptosis. We investigated the mechanisms by which PTH inhibits ERK1/2 activity in osteoblastic UMR 106‐01 cells. Treatment with PTH significantly inhibited phosphorylated ERK1/2 between 5 and 60 min. Transient transfection of cells with a cDNA encoding MAPK phosphatase‐1 (MKP‐1) resulted in 30–40% inhibition of pERK1/2; however MKP‐1 protein levels were only significantly stimulated by PTH after 30 mins, suggesting another mechanism for the early phase of pERK1/2 inhibition. The active upstream kinase c‐Raf phosphorylation at serine 338 (ser³³⁸) was significantly inhibited by PTH treatment within 5 min and transfection of the cells with constitutively‐active c‐Raf blocked PTH inhibition of pERK1/2. Inhibition of pERK1/2 and phosphor‐c‐Raf were seen when cells were treated with PTH(1‐34) or PTH(1‐31) analogues that stimulate cAMP, but not with PTH(3‐34), PTH(7‐34) or PTH(18‐48) that do not stimulate cAMP. Stimulation of the cells with forskolin or 8BrcAMP also inhibited pERK1/2 and c‐Raf.p338. Our results suggest that rapid PTH inhibition of ERK1/2 activity is mediated by PKA dependent inhibition of c‐Raf activity and that stimulation of MKP‐1 may contribute to maintaining pERK1/2 inhibition over prolonged time. Copyright © 2009 John Wiley & Sons, Ltd.     

Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0-40 min) of LLCP-K(1) cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression.     

Quantitative phosphoproteomic analysis reveals γ‐bisabolene inducing p53‐mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation

γ‐Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti‐proliferative activities against human oral squamous cell carcinoma (OSCC). γ‐Bisabolene activated caspases‐3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9‐22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ‐bisabolene was identified using TiO2‐PDMS plate and LC‐MS/MS, then confirmed using Western blotting and real‐time RT‐PCR assays. Phosphoproteome profiling revealed that γ‐bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein–protein interaction network analysis proposed the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in γ‐bisabolene‐induced apoptosis. Subsequent assays indicated γ‐bisabolene eliciting p53 acetylation that enhanced the expression of p53‐regulated apoptotic genes. PP1 inhibitor‐2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ‐bisabolene‐treated Ca9‐22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ‐bisabolene‐induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in mitochondria‐mediated apoptosis of γ‐bisabolene‐treated cells. This study demonstrated γ‐bisabolene displaying potent anti‐proliferative and apoptosis‐inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ‐bisabolene‐induced apoptosis. The novel insight could be useful for developing anti‐cancer drugs.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Oligomeric aggregates of amyloid β peptide 1–42 activate ERK/MAPK in SH-SY5Y cells via the α7 nicotinic receptor

The production and aggregation of amyloid β peptides (Aβ) has been linked to the development and progression of Alzheimer's disease. It is apparent that the various structural forms of Aβ can affect cell signalling pathways and the activity of neurons differently. In this study, we investigated the effects of oligomeric and fibrillar aggregates of Aβ 1–42 (Aβ42) and non-aggregated peptide upon activation of the ERK/MAPK signalling pathway. In SH-SY5Y cells, acute exposure to oligomeric Aβ42 led to phosphorylation of ERK1/2 at concentrations as low as 1 nM and up to 100 nM. These changes were detected as early as 5 min following exposure to 100 nM oligomeric Aβ42, reaching a maximum level after 10 min. Phosphorylation of ERK1/2 subsequently declined to and remained at basal levels after 30 min to 2 h of exposure. Fibrillar aggregates of Aβ42 did not significantly induce phosphorylation of ERK1/2 and non-aggregated Aβ42 did not activate the pathway. The effects of oligomeric Aβ42 to increase ERK phosphorylation above basal levels were inhibited by MLA, a specific antagonist of the α7 nAChR. U0126, an inhibitor of MEK, the upstream activator of ERK1/2, completely blocked induction of ERK1/2 phosphorylation. Oligomeric aggregates of Aβ42 are the principal structural form of the peptide that activates ERK/MAPK in SH-SY5Y cells and these effects are mediated by the α7 nAChR.     

Heterodimerization of apelin receptor and neurotensin receptor 1 induces phosphorylation of ERK1/2 and cell proliferation via Gαq‐mediated mechanism

Dimerization of G protein‐coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin‐converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C‐terminal residues of vasoactive peptides including apelin‐13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co‐immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK_1/2 activation and increased proliferation via activation of Gq α‐subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK_1/2 activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.     

Regulation of ERK1/2 activity upon contact inhibition in fibroblasts

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.     

Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.