Dopaminergic differentiation of the Nurr1‐expressing immortalized mesencephalic cell line CSM14.1<i>in vitro</i>

Haas, Stefan; Wree, Andreas

doi:10.1046/j.1469-7580.2002.00072.x

Cited by 29 publications

(34 citation statements)

References 44 publications

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“…CSM14.1 cells are derived from the ventral mesencephalic region of an E14 rat, and immortalized with the temperaturesensitive Large T antigen. 36 When cultured at 331C (permissive temperature), CSM14.1 cells express the large T antigen, proliferate, have a flat neuroepitheliallike morphology and express the neural stem cell marker nestin. 36 When transferred to 391C, the temperature-sensitive large T antigen is inactivated and proliferation ceases.…”

Section: Discussionmentioning

confidence: 99%

“…36 When cultured at 331C (permissive temperature), CSM14.1 cells express the large T antigen, proliferate, have a flat neuroepitheliallike morphology and express the neural stem cell marker nestin. 36 When transferred to 391C, the temperature-sensitive large T antigen is inactivated and proliferation ceases. Moreover, CSM14.1 cells acquire a neuron-like morphology, extend neurites, and differentiate into neuronal cells expressing the dopaminergic markers Nurr1, TH and ALDH2.…”

Section: Discussionmentioning

confidence: 99%

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Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

et al. 2007

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Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominantnegative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways. Mitochondria can acquire a broad range of morphologies, from a punctuate shape to a tubular appearance or even extended networks. Several proteins, most of them belonging to the family of large GTPases, have been identified that energy-dependently regulate mitochondrial morphology and subcellular distribution by promoting either fission (Fis1, dynamin-related protein 1 (Drp1), MTP18) or fusion of mitochondria (Mfn1/2 and Opa1). 1-3 As a potential upstream regulatory factor, the actin cytoskeleton has been shown to be a prerequisite for mitochondrial fission, possibly by its influence on Drp1 recruitment to mitochondria. 4 Apoptotic release of the mitochondrial cytochrome c and other proapoptotic mitochondrial proteins results in formation of the so-called apoptosome, which in turn activates downstream effector caspases with death executing function. 5 This intrinsic, mitochondrial death pathway is relevant for most forms of neuronal apoptosis, in contrast to extrinsic, for example death receptor mediated, activation of programmed cell death. 6 However, despite the contribution of mitochondrial dysfunction specifically to the demise of neurons, there is only scarce knowledge about mitochondrial fission during neuronal apoptosis, nor about its functional contribution to neuronal cell death. Furthermore, little is known about upstream regulatory pathways for mitochondrial morphology in general.Cyclin-dependent kinase 5 (CDK5) is a member of the CDK family, but, distinct from other CDKs, it does not participate in cell cycle regulation. 7 Instead, CDK5 is physiologically implicated in cytoskeletal functions, as well as regulation of membrane turnover and morphology, for example cell migration or endocytosis. Moreover, it is involved in neuronspecific functions including, for example, synaptic plasticity, axonal outgrowth or transmitter release. [8][9][10][11] In neurological diseases, deregulated CDK5 turned out to be an apical instigator of neuronal cell death cascades. Amyloid-toxicity was shown to induce calpain-mediated cleavage of the CDK5 activators p35 or p39 to p25 and p29. This leads to redistribution and overactivation of CDK5 after its association...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

et al. 2007

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“…CSM14.1 is a rat striatal neuroprogenitor cell line that was established by immortalization using a temperature-sensitive variant of SV40 large T antigen (11). For convenience, and because transient reductions in temperature that might be associated with large screening experiments could restore large T activity, we developed our HTS assay using undifferentiated CSM14.1 cells, with the plan to then confirm hits using differentiated cells.…”

Section: Cell-based Screen For Inhibitors Of Er Stress-induced Cellmentioning

confidence: 99%

Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1

Kim

Shu

et al. 2009

Journal of Biological Chemistry

124

104

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The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.

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“…The rat nigrostriatal cell line CSM14.1, immortalized by introduction of the temperature-sensitive simian virus 40 large T antigen, 36 was cultured in complete DMEM supplemented with 10% heatinactivated fetal calf serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin at 321C in 5% CO 2 . For transfection, cells were seeded on poly-L-ornithine/laminin (Sigma, Cölbe, Germany)-coated coverslips (12 mm diameter) at a density of 15 000 cells/cm 2 .…”

Section: Methodsmentioning

confidence: 99%

Mutant SOD1 detoxification mechanisms in intact single cells

et al. 2007

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Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cellby-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS. Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease caused by the selective loss of motoneurons. 1 While ALS mostly occurs sporadically, 5-10% of cases are hereditary. About 20% of familial and few sporadic cases show point or frameshift mutations within the superoxide dismutase 1 (SOD1) gene. 2 The principle underlying mutant superoxide dismutase 1 (mtSOD1) toxicity seems to be a toxic gain -rather than loss -of function, since symptomatic mtSOD1-transgenic mice still express wild-type (wt), SOD1, some SOD1 mutants retain their enzymatic activity, 2,3 and neither knockout of endogenous SOD1 nor transgenic overexpression of wtSOD1 affect the course of disease in mtSOD1-transgenic mice. 4 Nonetheless, the downstream events in mtSOD1-mediated motoneuron death have not yet been resolved, although several hypotheses have been proposed that are based on properties that are specific for mtSOD1: altered protein folding, decreased solubility, and the propensity to form cytoplasmic aggregates. 5-10 One of the major hypotheses states that misfolded mtSOD1, through binding, depletes neuroprotective chaperones. 5-7 Accordingly, recombinant mtSOD1 inhibited chaperone activity in vitro. 11 Moreover, expression of Hsp70, Hsp27, or Hsp40 decreased aggregation of mtSOD1 and protected against mtSOD1 toxicity in cell lines and primary motoneurons. 6,12 However, overexpression of Hsp70 alone did not improve motoneuron survival in mtSOD1-transgenic mice. 13 This is possibly explained by a requirement, in vivo, for correction of further cellular functions or the combined action of different chaperones in the prevention of motoneuron cell death. Indeed, arimoclomol treatment, known to upregulate both Hsp70 and Hsp90 in the spinal cord, extended the lifespan of SOD1 ...

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Dopaminergic differentiation of the Nurr1‐expressing immortalized mesencephalic cell line CSM14.1in vitro

Cited by 29 publications

References 44 publications

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1

Mutant SOD1 detoxification mechanisms in intact single cells

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