Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

Collins, A. J.; Larson, Maureen K.; Pfaff, Jilleen E.; Ishmael, Jane E.

doi:10.1016/j.toxlet.2007.04.004

Cited by 4 publications

(3 citation statements)

References 43 publications

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“…Cells were then collected by the addition of serum-free DMEM (40 µL) mixed with Trypan Blue and the viable cells counted. LDH activity was measured as previously described [9] using a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

et al. 2013

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Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

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Section: Methodsmentioning

confidence: 99%

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

et al. 2013

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“…Notably, rat and mouse CGCs-albeit there is still some controversy about this latter species (Collins et al, 2007)-die when maintained in vitro with physiological [K + ] (3.5-5 mM) under nondepolarization conditions, and, to avoid this, are usually cultivated in high [K + ] (25 mM) under sustained depolarization. However, observations on isolated CGCs (Sato et al, 2005;Nakanishi and Okazawa, 2006) have demonstrated that these neurons develop and become mature in a temporally regulated manner under physiological [K + ] e , whereas elevated [K + ] e blocks the process of differentiation at a stage of immaturity.…”

Section: Introductionmentioning

confidence: 99%

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Lossi

Gambino

Ferrini

et al. 2009

Developmental Neurobiology

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Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

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“…We show here that mouse NGCCs are susceptible to 5-10 µM A β 1-40 only when maintained in 25 mM KCl. Interestingly, it was previously demonstrated that, differently from rat, isolated mouse CGCs can also survive in vitro in the presence of 5 mM KCl [34]. However, in both species, these neurons develop and become mature in a temporally regulated manner under physiological KCl, whereas elevated KCl blocks their differentiation during immaturity [35][36][37][38][39].…”

Section: Response Of Ngccs To Aβ 1-40mentioning

confidence: 99%

Context-Dependent Toxicity of Amyloid-β Peptides on Mouse Cerebellar Cells

Alasia

Aimar

Merighi

et al. 2012

JAD

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Alzheimer's disease (AD) is the major cause of dementia in old people. AD pathology is characterized by amyloid-β (Aβ) deposits in several regions of the brain, and links have been hypothesized between Aβ toxicity and apoptosis. Cerebellar granule cells (CGCs) have been widely used as in vitro tools for molecular studies correlating apoptosis with AD, although the cerebellum is a relatively spared area of the brain in vivo. We have used mixed neuronal-glial cerebellar cultures (NGCCs) and organotypic cerebellar cultures (OCCs) obtained from postnatal mice to assess the toxic effect of the Aβ oligomer 1-40 (Aβ1-40) after propidium iodide uptake in vitro. Our results demonstrate that NGCCs, which are primarily composed of CGCs, are resistant to Aβ1-40 challenge (5-10 μM) when cultured in physiological (5 mM) extracellular KCl ([K+]e) concentrations, i.e., in a condition in which CGCs undergo full maturation. Conversely, when 10 μM Aβ1-40 is given to NGCCs cultured in elevated (25 mM) [K+]e (and thus maintained in an immature state), there is a statistically significant increase in cell death. Cell death is by apoptosis, as demonstrated by ultrastructural examination. OCCs are resistant to Aβ challenge in any of the conditions tested (variation of [K+]e, presence or absence of serum, or addition of the neprilysin blocker phosphoramidon). Altogether these observations lead us to conclude that cerebellar cells in an organotypic context may be less susceptible to damage by Aβ, raising the question whether isolated CGCs are a reliable assay in drug discovery studies of AD.

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Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

Cited by 4 publications

References 43 publications

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Context-Dependent Toxicity of Amyloid-β Peptides on Mouse Cerebellar Cells

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