Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates

McCarthy, Ken D.; Partlow, Lester M.

doi:10.1016/0006-8993(76)90962-8

Cited by 131 publications

(47 citation statements)

References 40 publications

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“…To challenge a function of caspasetruncated RET as a cadherin accessory protein improving cadherin-mediated adhesion in sympathetic neurons, we nucleofected different Myc-tagged RET constructs in dissociated SCG neurons, and we analyzed neurons by immunofluorescence after 48 h of culture at low density. As it usually occurs, somas of dissociated sympathetic neurons seeded for culture tend to reaggregate with time (25). However, nucleofection of caspasetruncated RET accelerated this tendency, and at 48 h we observed a higher number of aggregated neuronal somas (Fig.…”

Section: Resultssupporting

confidence: 52%

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

Cabrera¹,

Bouzas-Rodríguez²,

Tauszig-Delamasure³

et al. 2011

Journal of Biological Chemistry

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RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/ survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.A limited number of growth factors and their cognate receptors are required to convey the development of extraordinarily complex biological structures. This suggests that cells maximize their molecular resources. It is then tempting to assume that selection of multitasked receptors has been favored during evolution. The RET tyrosine kinase receptor is a good example for the integration of multiple functions in a single protein. RET mediates many different activities during development but also through adulthood. It has been described that RET induces survival, differentiation, proliferation, migration, or axonal guidance via activation of canonical signaling pathways like PI3K and MAPK (for review, see Ref. 1). This ability to promote different biological effects is partially due to an exquisitely coordinated and regulated relationship between RET and its ligands. RET can be activated by the GDNF 5 family ligands (GFLs), a family composed of four members: GDNF, neurturin, artemin, and persephin. However, RET cannot directly bind any of these ligands but requires the presence of the accessory proteins GDNF family receptor ␣1-4 (GFR␣1-4) for its tyrosine kinase catalytic activation (2). The general view is that GFL binding to GFR␣/RET triggers RET dimerization, autophosphorylation, and tyrosine kinase activation of RET under physiological conditions, although RET tyrosine kinase activity can be activated in a ligand-independent manner under some pathological circumstances (3).Although many tyrosine kinase receptors can activate multiple pathways to promote different biological effects, RET is yet an atypical tyrosine kinase receptor. RET belongs to the cadherin superfamily, and it has been sugge...

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Section: Resultssupporting

confidence: 52%

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

Cabrera¹,

Bouzas-Rodríguez²,

Tauszig-Delamasure³

et al. 2011

Journal of Biological Chemistry

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“…For 6-well plate studies (Costar 9.5 cm 2 /well) wells were pre-coated with 1.0 ml/well of polyornithine and 1.0 ml/well of laminin at the concentrations listed above. Highly purified neuronal preparations were obtained by incubating DRG cell mixtures in 100-mm diameter plastic tissue culture dishes coated with type I rat tail collagen (Collaborative Biomedical Products) (35). Cells were incubated for 2.5 h at 37°C in 5% CO 2 , 95% air and shaken for 30 s every 10 -15 min, resulting in a supernatant containing Ͼ95% neurons.…”

Section: Methodsmentioning

confidence: 99%

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Xie

Tisi

Yeo

et al. 2000

Journal of Biological Chemistry

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Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite-but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.Nerve growth factor (NGF) 1 acts via TrkA and p75 receptors to regulate neuronal survival, promote neurite outgrowth, and up-regulate certain neuronal functions such as mediation of pain and inflammation (1-5). These actions suggest that NGF agonists or antagonists might be useful in regulating these processes (6 -8). Factors limiting therapeutic applications of the NGF protein include restricted penetration of the central nervous system and the poor medicinal properties characteristic of most proteins (9, 10). The development of small molecule mimetics with favorable chemical properties that function as agonists or antagonists that mimic or inhibit NGF functions in the appropriate biological context will be critical in advancing potential in vivo applications of NGF. Moreover, in settings in which NGF might contribute to neuronal death, pain, or inflammatory mechanisms, NGF antagonists may be particularly relevant. Creation of single domain NGF mimetics will also constitute a powerful approach for linking specific NGF domains with specific patterns of intracellular signal transduction.A NGF mimetic (agonist or antagonist) would be expected to contain structural determinants of one or more NGF active sites that interact with NGF receptors. Multiple techniques have been used to deduce which domains of the NGF protein interact with NGF receptors (11). A peptide mapping approach in which synthetic peptides with sequences corresponding to specific NGF regions were tested for their ability to inhibit NGF activity pointed to residues 29 -35 as a key active ...

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“…These were the patch-clamp, developed by Bert Sakmann and Ervin Neher in collaboration with Owen Hamill, Fred Sigworth and Alan Marty [33]; the florescent calcium indicators invented by Roger Tsien [83,84] and the technique of preparation and maintenance of glial cell cultures [57,58]. These techniques used alone or in combination were instrumental for many fundamental discoveries, which completely change our comprehension of the importance of glial cells in the nervous system.…”

Section: The Turn Of the Tide-identification Of Glial Excitationmentioning

confidence: 99%

Patching the glia reveals the functional organisation of the brain

Verkhratsky

2006

Pflugers Arch - Eur J Physiol

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The neuroglia was initially conceived by Rudolf Virchow as a non-cellular connective tissue holding neurones together. In 1894, Carl Ludwig Schleich proposed a hypothesis of fully integrated and interconnected neuronalglial circuits as a substrate for brain function. This hypothesis received direct experimental support only hundred years later, after several physiological techniques, and most notably the patch-clamp method, were applied to glial cells. These experiments have demonstrated the existence of active and bi-directional neuronal-glial communications, integrating neuronal networks and glial syncytium into one functional circuit. The data accumulated during last 15 years prompt rethinking of the neuronal doctrine towards more inclusive concept, which regards both neurones and glia as equally responsible for information processing in the brain.

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Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates

Cited by 131 publications

References 40 publications

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Patching the glia reveals the functional organisation of the brain

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