Occurrence of Two Types of Secretory Vesicles in the Human Neuroblastoma SH‐SY5Y

Goodall, Anna R.; Danks, Kirsty; Walker, John H.; Ball, Stephen G.; Vaughan, Peter F. T.

doi:10.1046/j.1471-4159.1997.68041542.x

Cited by 46 publications

(35 citation statements)

References 28 publications

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“…Neurites and their boutons responded to receptor activation ( Fig. 1e) and have been proposed to provide release sites [39,40]. We therefore probed the effect of A 2A -receptor activation on noradrenaline release.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

Ibrišimović

Drobny

Yang

et al. 2012

Purinergic Signalling

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Neuroblastoma SH-SY5Y (SH) cells endogenously express A 2A adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A 2A -receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A 2A -receptor antagonist, XAC, as well as an inhibitor of cAMPdependent protein kinase A (PKA), H89, depressed agonistdependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A 2A -receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltagedependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A 2A -receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.

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Section: Resultsmentioning

confidence: 99%

“…1b). Probably, release sites are present on SH-cell neurites, which were found to be rich in vesicles and proteins of the vesicle-fusion apparatus [39,40]. Thus, A 2A -receptors may be located close to release sites and, in addition, they meet the criteria for docking to the 'presynaptic' active zone.…”

Section: Discussionmentioning

confidence: 99%

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

Ibrišimović

Drobny

Yang

et al. 2012

Purinergic Signalling

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“…In SH-SY5Y cells, it has been suggested that two types of secretory vesicles exist, small synaptic-like vesicles (SSLVs) and the large dense core vesicles (LDCVs) (Goodall et al, 1997). LDCVs can be followed with a specific marker, secretogranin II, and distinguished from lighter SSLVs with synaptophysin, which is present only in light vesicles.…”

Section: Intravesicular Localization Of ␣-Synmentioning

confidence: 99%

Intravesicular Localization and Exocytosis of α-Synuclein and its Aggregates

Lee¹,

Patel

Lee³

2005

J. Neurosci.

745

703

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␣-Synuclein (␣-syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of ␣-syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, ␣-syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized ␣-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular ␣-syn is present in the lumen of vesicles. Importantly, the intravesicular ␣-syn is more prone to aggregation than the cytosolic protein, and aggregated forms of ␣-syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated ␣-syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of ␣-syn and might also contribute to pathological function of this protein.

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“…For these studies, we used human neuroblastoma SH-SY5Y cells, a well characterized dopaminergic cell line that expresses endogenous syntaxin 1 and exhibits SNARE-mediated neurotransmitter release (54,55). Overexpression of Staring in SH-SY5Y cells significantly enhanced the ubiquitination of endogenous syntaxin 1, as indicated by the appearance of a high molecular weight smear containing Myc-ubiquitin-modified syntaxin 1 (Fig.…”

Section: Figmentioning

confidence: 99%

Staring, a Novel E3 Ubiquitin-Protein Ligase That Targets Syntaxin 1 for Degradation

Chin

Vavalle

Lian

2002

Journal of Biological Chemistry

111

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Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway.Modulation of protein degradation is a major mechanism by which cells regulate the expression levels of specific proteins and consequently the cellular processes that these proteins participate in (1, 2). The ubiquitin-proteasome pathway plays a crucial role in the degradation of proteins involved in a variety of cellular processes, including differentiation, proliferation, and apoptosis. However, the role of the ubiquitin-proteasome pathway in the degradation of presynaptic proteins remains poorly characterized, despite the presence of ubiquitin at nerve terminals (3-5). In the ubiquitin-proteasome pathway, substrates are marked for degradation by covalent linkage to ubiquitin. The ubiquitinated substrate proteins are then recognized and degraded by the 26 S proteasome (1, 2, 6). Ubiquitination involves a highly specific enzyme cascade in which ubiquitin is first activated by an E1 1 ubiquitin-activating enzyme and then transferred to an E2 ubiquitin-conjugating enzyme and finally ligated to the substrate by an E3 ubiquitin-protein ligase (1,7,8). The E3 ubiquitin-protein ligase plays an essential role in determining the specificity of ubiquitination and subsequent protein degradation. Consistent with this role, it is estimated that an organism such as a human contains over 100 E3 ubiquitin ligases, in contrast to a single E1 ubiquitin-activating enzyme and about a dozen E2 ubiquitin-conjugating enzymes (9). Despite the importance of E3 ubiquitin-protein ligases in specific protein degradation and the estimated presence of more than 100 E3 ligases in the human genome, only a few E3 ligases have been characterized at the molecular level.Syntaxin 1 is a neuronal membrane protein that was originally identified as a binding partner for synaptotagmin and the N-type calcium channel (10 -12). It is well established that syntaxin 1 functions as a synaptic t-SNARE to mediate synaptic vesicle exocytosis at nerve terminals (13)(14)(15). Syntaxin 1 appears early during embryonic development (16, 17), and its expression level is dramatically up-regulated during synapse formation and brain maturation (16 -19). Regulation of syntaxin 1 levels may contribute to the mechanism unde...

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Occurrence of Two Types of Secretory Vesicles in the Human Neuroblastoma SH‐SY5Y

Cited by 46 publications

References 28 publications

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

Intravesicular Localization and Exocytosis of α-Synuclein and its Aggregates

Staring, a Novel E3 Ubiquitin-Protein Ligase That Targets Syntaxin 1 for Degradation

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