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Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.
Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this family's GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and β2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.
Copyright © 2014 Elsevier Inc. All rights reserved.
Fluorescent dyes have been designed for internal cellular component specificity and have been used extensively in the scientific community as a means to monitor cell growth, location, morphology, and viability. However, it is possible that the introduction of these dyes influences the basal function of the cell and, in turn, the results of these studies. Electrochemistry provides a noninvasive method for probing the unintended cellular affects of these dyes. The multianalyte microphysiometer (MAMP) is capable of simultaneous electrochemical measurement of extracellular metabolites in real-time. In this study, analytes central to cellular metabolism, glucose, lactate, oxygen, as well as extracellular acidification were monitored to determine the immediate metabolic effects of nuclear stains, including SYTO, DAPI dilactate, Hoechst 33342, and FITC dyes upon the pheochromocytoma PC-12 cells and RAW 264.7 macrophages. The experimental results revealed that the SYTO dye 13 significantly decreased glucose and oxygen consumption and increased extracellular acidification and lactate production in both cell lines, indicating a shift to anaerobic respiration. No other dyes caused significantly definitive changes in cellular metabolism upon exposure. This study shows that fluorescent dyes can have unintended effects on cellular metabolism and care should be taken when using these probes to investigate cellular function and morphology.
Multianalyte microphysiometry, a real-time instrument for simultaneous measurement of metabolic analytes in a microfluidic environment, was used to explore the effects of cholera toxin (CTx). Upon exposure of CTx to PC-12 cells, anaerobic respiration was triggered, measured as increases in acid and lactate production and a decrease in the oxygen uptake. We believe the responses observed are due to a CTx-induced activation of adenylate cyclase, increasing cAMP production and resulting in a switch to anaerobic respiration. Inhibitors (H-89, brefeldin A) and stimulators (forskolin) of cAMP were employed to modulate the CTx-induced cAMP responses. The results of this study show the utility of multianalyte microphysiometry to quantitatively determine the dynamic metabolic effects of toxins and affected pathways.
Ketamine is an NMDA antagonist and dissociative anesthetic that has been shown to display rapid acting and prolonged antidepressant activity in small-scale human clinical trials. Ketamine also binds to σ receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine the involvement of σ receptors in the antidepressant-like actions of ketamine. Competition binding assays were performed to assess the affinity of ketamine for σ(1) and σ(2) receptors. The antidepressant-like effects of ketamine were assessed in vitro using a neurite outgrowth model and PC12 cells, and in vivo using the forced swim test. The σ receptor antagonists, NE-100 and BD1047, were evaluated in conjunction with ketamine in these assays to determine the involvement of σ receptors in the antidepressant-like effects of ketamine. Ketamine bound to both σ(1) and σ(2) receptors with μM affinities. Additionally, ketamine potentiated NGF-induced neurite outgrowth in PC12 cells and this effect was attenuated in the presence of NE-100. Ketamine also displayed antidepressant-like effects in the forced swim test; however, these effects were not attenuated by pretreatment with NE-100 or BD1047. Taken together, these data suggest that σ receptor-mediated neuronal remodeling may contribute to the antidepressant effects of ketamine.
Copyright Â© 2011 Elsevier B.V. and ECNP. All rights reserved.
Nerve growth factor (NGF) induces autophosphorylation and downstream progrowth and prosurvival signaling from the receptor tyrosine kinase TrkA. Overexpression or activating mutation of TrkA has been described in human acute myeloid leukemia cells. In the present study, we show the chaperone association of TrkA with heat shock protein 90 (hsp90) and the inhibitory effect of the hsp90 inhibitor, 17-DMAG, on TrkA levels and signaling in cultured and primary myeloid leukemia cells. Treatment with 17-DMAG disrupted the binding of TrkA with hsp90 and the cochaperone cdc37, resulting in polyubiquitylation, proteasomal degradation, and depletion of TrkA. Exposure to 17-DMAG inhibited NGF-induced p-TrkA, p-AKT, and p-ERK1/2 levels, as well as induced apoptosis of K562, 32D cells with ectopic expression of wild-type TrkA or the constitutively active mutant Delta TrkA, and of primary myeloid leukemia cells. Additionally, 17-DMAG treatment inhibited NGF-induced neurite formation in the rat pheochromocytoma PC-12 cells. Cotreatment with 17-DMAG and K-252a, an inhibitor of TrkA-mediated signaling, induced synergistic loss of viability of cultured and primary myeloid leukemia cells. These findings show that TrkA is an hsp90 client protein, and inhibition of hsp90 depletes TrkA and its progrowth and prosurvival signaling in myeloid leukemia cells. These findings also support further evaluation of the combined activity of an hsp90 inhibitor and TrkA antagonist against myeloid leukemia cells.
(c) 2010 AACR.
Free radical-mediated lipid peroxidation has been implicated in the pathogenesis of various diseases. Lipid peroxidation products are cytotoxic and they modify proteins and DNA bases, leading eventually to degenerative disorders. Various synthetic antioxidants have been developed and assessed for their capacity to inhibit lipid peroxidation and oxidative stress induced by free radicals. In this study, the capacity of novel 6-amino-2,4,5-trimethyl-3-pyridinols for scavenging peroxyl radicals, inhibiting plasma lipid peroxidation in vitro, and preventing cytotoxicity induced by glutamate, 6-hydroxydopamine, 1-methyl-4-phenylpyridium (MPP(+) ), and hydroperoxyoctadecadienoic acid was assessed. It was found that they exerted higher reactivity toward peroxyl radicals and more potent activity for inhibiting the above oxidative stress than alpha-tocopherol, the most potent natural antioxidant, except against the cytotoxicity induced by MPP(+). These results suggest that the novel 6-amino-3-pyridinols may be potent antioxidants against oxidative stress.
Copyright 2010 Elsevier Inc. All rights reserved.
Alpha-synuclein (alphaSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding alphaSyn causes early onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of alphaSyn. Several studies have reported evidence that alphaSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of alphaSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of alphaSyn, we sought to extend existing reports on this interaction. Using purified proteins, we tested the ability of alphaSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether alphaSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between alphaSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by alphaSyn in any of these systems. We propose that the previously reported effects of alphaSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with alphaSyn overexpression in cells, but are not likely due to a specific and direct interaction between alphaSyn and PLD.
The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.