Biomedical subjects

Andreas Wyttenbach

Publications and source records attributed to Andreas Wyttenbach.

10 recordsLinked to original sources

PubMed2006

The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons.

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.

Animals↗

PubMed2006

Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant.

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.

Acetylcysteine↗

PubMed2006

Huntingtin inclusion bodies are iron-dependent centers of oxidative events.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

Acetylcysteine↗

PubMed2005

Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins.

We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with (32)Pi, whereas cells in which the kinase is active are labeled with (33)Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both (32)P- and (33)P-labeled proteins; the kinase-specific spots are revealed because of (33)P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic (33)P-labeled proteins while allowing the more energetic (32)P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons.

Acetates↗

PubMed2005

Mutually exclusive subsets of BH3-only proteins are activated by the p53 and c-Jun N-terminal kinase/c-Jun signaling pathways during cortical neuron apoptosis induced by arsenite.

The c-Jun N-terminal protein kinase (JNK)/c-Jun and p53 pathways form distinct death-signaling modules in neurons that culminate in Bax-dependent apoptosis. To investigate whether this signaling autonomy is due to recruitment of particular BH3-only proteins, we searched for a toxic signal that would activate both pathways in the same set of neurons. We show that arsenite activates both the JNK/c-Jun and p53 pathways in cortical neurons, which together account for >95% of apoptosis, as determined by using the mixed-lineage kinase (JNK/c-Jun) pathway inhibitor CEP11004 and p53-null mice. Despite the coexistence of both pathways in at least 30% of the population, Bim mRNA and protein expression was increased only by the JNK/c-Jun signaling pathway, whereas Noxa and Puma mRNA and Puma protein expression was entirely JNK/c-Jun independent. About 50% of Puma/Noxa expression was p53 dependent, with the remaining signal being independent of both pathways and possibly facilitated by arsenite-induced reduction in P-Akt. However, functionally, Puma was predominant in mediating Bax-dependent apoptosis, as evidenced by the fact that more than 90% of apoptosis was prevented in Puma-null neurons, although Bim was still upregulated, while Bim- and Noxa-null neurons died similarly to wild-type neurons. Thus, the p53 and JNK/c-Jun pathways can activate mutually exclusive subclasses of BH3-only proteins in the same set of neurons. However, other factors besides expression may determine which BH3-only proteins mediate apoptosis.

Animals↗

PubMed2004

Aggravation of necrotic death of glucose-deprived cells by the MEK1 inhibitors U0126 and PD184161 through depletion of ATP.

The extracellular-regulated kinases (ERK) modulate cell proliferation and survival in response to several different stimuli and are therefore important drug targets. ERKs are activated by the dual phosphorylation kinase MEK1 and MEK1 inhibitors PD98059, U0126 and CI-1040 are now widely used to inhibit ERKs in cell and animal studies. In an analysis of ERK functions in astrocytes we found that PD98059 (100microM) failed to inhibit ERK phosphorylation but U0126 (50microM) inhibited ERK phosphorylation to approximately 80%. Surprisingly, U0126 also caused profound depletion of ATP in glucose-deprived cells, leading to death by necrosis. Since glucose-deprived cells depend mainly on mitochondrial ATP-synthase for ATP production, we tested whether U0126 or PD184161, a derivative of CI-1040, might inhibit ATP synthase activity, using 143B(Rho0) cells (which lack a functional F0 subunit) to further parse this effect. We found that the F1F0ATPase activity extracted from U0126- or PD184161-treated parental 143B cells or astrocytes was indeed inhibited by >or=80% suggesting a covalent change in the enzyme. However, F1F0ATPase activity extracted from similarly treated 143B(Rho0) cells was spared. Because F1F0ATPase activity in isolated mitochondria was not inhibited directly, we propose that U0126 and PD184161 inhibit ATP-synthase via an indirect action on F0. The MEK1 inhibitors also induced necrosis of other glucose-deprived cell types including primary neurons at the same concentrations required for inhibition of ERK phosphorylation. Thus, the MEK1/ERK signalling pathway may modulate ATP synthase function, and its inhibition may cause cells unable to perform glycolysis to die by necrosis.

Adenosine Triphosphate↗

PubMed2004

Role of heat shock proteins during polyglutamine neurodegeneration: mechanisms and hypothesis.

A common feature of many neurodegenerative diseases, including Alzheimer's and Parkinsons's disease, the prion disorders, and the CAG repeat polyglutamine (polyQ) diseases, is the occurrence of protein aggregates within or outside of nerve cells. Molecular chaperones such as heat shock proteins (HSPs) have been proposed to play a critical role in preventing the accumulation of misfolded proteins that lead to the deposition of aggregates during pathology. This article focuses on the role of HSPs during polyQ pathologies, which include Huntington's disease, spinal and bulbar muscular atrophy, dentatorubral and pallidoluysian atrophy, and several forms of spinocerebellar ataxia. Recently, unifying mechanisms that are involved during polyQ disease have emerged, such as abnormal transcription, impaired degradation systems, and interference of a polyQ expansion with neuronal survival and death-signaling pathways like the activation of caspases and kinases. This article reviews recent studies that point to the involvement of these mechanisms during polyQ pathology and discusses how HSPs can interfere with such processes by paying special attention to HSPs as modulators of survival and death-signaling pathways.

Animals↗

PubMed2003

Expression of mutant huntingtin blocks exocytosis in PC12 cells by depletion of complexin II.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. We reported recently that complexin II, a protein involved in neurotransmitter release, is depleted from both the brains of mice carrying the HD mutation and from the striatum of post mortem HD brains. Here we show that this loss of complexin II is recapitulated in PC12 cells expressing the HD mutation and is accompanied by a dramatic decline in Ca2+-triggered exocytosis of neurotransmitter. Overexpression of complexin II (but not complexin I) rescued exocytosis, demonstrating that the decline in neurotransmitter release is a direct consequence of complexin II depletion. Complexin II depletion in the brain may account for some of the abnormalities in neurotransmission associated with HD.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Modulation of polyglutamine-induced cell death by genes identified by expression profiling.

The majority of triplet-repeat diseases are caused by mutated genes with an extended polyglutamine tract, exemplified by Huntington's disease (HD). In order to model HD pathogenesis in a controlled system, we developed stable PC12 cell lines that express exon 1 fragments of the huntingtin gene with 23 or 74 polyglutamines driven by an inducible doxycycline (dox)-sensitive promoter (HD-23Q or HD-74Q). We aimed to identify early perturbations induced by the mutation by studying expression levels of 1824 genes at 0, 5, 10 and 18 hours after induction, using adaptor-tagged competitive PCR (ATAC-PCR). At these time points, the cells show no appreciable death or mitochondrial impairment and very low inclusion levels. A total of 126 genes, including 69 known genes, exhibited statistically significant alterations in the HD-74Q cell lines but no changes in the HD-23Q lines. We tested 11 of these genes for their abilities to modulate polyglutamine-induced cell death in transiently transfected cell models. Five genes [glucose transporter 1 (Glut1), phosphofructokinase muscle isozyme (Pfkm), prostate glutathione-S -transferase 2 (Gstm2), RNA-binding motif protein 3 (Rbm3) and KRAB-A interacting protein 1 (Krip-1)] significantly suppressed cell death in both neuronal precursor and non-neuronal cell lines, suggesting that these transcriptional changes were relevant to polyglutamine pathology. The efficient recovery of functionally relevant genes supports the utility of gene expression profiling for discovering pathways related to pathogenesis, and the importance of analyzing molecular events in the early stages of disease.

Animals↗

PubMed2002

Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin.

Neuronal loss and intraneuronal protein aggregates are characteristics of Huntington's disease (HD), which is one of 10 known neurodegenerative disorders caused by an expanded polyglutamine [poly(Q)] tract in the disease protein. N-terminal fragments of mutant huntingtin produce intracellular aggregates and cause toxicity. Several studies have shown that chaperones suppress poly(Q) aggregation and toxicity/cell death, but the mechanisms by which they prevent poly(Q)-mediated cell death remain unclear. In the present study, we identified heat shock protein 27 (HSP27) as a suppressor of poly(Q) mediated cell death, using a cellular model of HD. In contrast to HSP40/70 chaperones, we showed that HSP27 suppressed poly(Q) death without suppressing poly(Q) aggregation. We tested the hypotheses that HSP27 may reduce poly(Q)-mediated cell death either by binding cytochrome c and inhibiting the mitochondrial death pathway or by protecting against reactive oxygen species (ROS). While poly(Q)-induced cell death was reduced by inhibiting cytochrome c (cyt c) release from mitochondria, protection by HSP27 was regulated by its phosphorylation status and was independent of its ability to bind to cyt c. However, we observed that mutant huntingtin caused increased levels of ROS in neuronal and non-neuronal cells. ROS contributed to cell death because both N-acetyl-L-cysteine and glutathione in its reduced form suppressed poly(Q)-mediated cell death. HSP27 decreased ROS in cells expressing mutant huntingtin, suggesting that this chaperone protects cells against oxidative stress. We propose that a poly(Q) mutation can induce ROS that directly contribute to cell death and that HSP27 is an antagonist of this process.

Animals↗