thapsigargin and Neurodegenerative-Diseases

The aim of our work was to study effect of antidepressant imipramine on both thapsigargin- and tunicamycin-induced ER stress and mitochondrial dysfunction in neuroblastoma SH-SY5Y cells. ER stress in SH-SY5Y cells was induced by either tunicamycin or thapsigargin in the presence or absence of imipramine. Cell viability was tested by the MTT assay. Splicing of XBP1 mRNA was studied by RT-PCR. Finally, expression of Hrd1 and Hsp60 was determined by Western blot analysis. Our findings provide evidence that at high concentrations imipramine potentiates ER stress-induced death of SH-SY5Y cells. The effect of imipramine on ER stress-induced death of SH-SY5Y cells was stronger in combination of imipramine with thapsigargin. In addition, we have found that treatment of SH-SY5Y cells with imipramine in combination of either thapsigargin or tunicamycin is associated with the alteration of ER stress-induced IRE1α-XBP1 signalling. Despite potentiation of ER stress-induced XBP1 splicing, imipramine suppresses both thapsigargin- and tunicamycin-induced expression of Hrd1. Finally, imipramine in combination with thapsigargin, but not tunicamycin, aggravates ER stress-induced mitochondrial dysfunction without significant impact on intracellular mitochondrial content as indicated by the unaltered expression of Hsp60. Our results indicate the possibility that chronic treatment with imipramine might be associated with a higher risk of development and progression of neurodegenerative disorders, in particular those allied with ER stress and mitochondrial dysfunction like Parkinson's and Alzheimer's disease.

Topics: Cell Death; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Endoribonucleases; Humans; Imipramine; Mitochondria; Neuroblastoma; Neurodegenerative Diseases; Protein Serine-Threonine Kinases; RNA Splicing; Signal Transduction; Thapsigargin; Tunicamycin; Ubiquitin-Protein Ligases; X-Box Binding Protein 1

Recent interest in astrocyte activation states has raised the fundamental question of how these cells, normally essential for synapse and neuronal maintenance, become pathogenic. Here, we show that activation of the unfolded protein response (UPR), specifically phosphorylated protein kinase R-like endoplasmic reticulum (ER) kinase (PERK-P) signaling-a pathway that is widely dysregulated in neurodegenerative diseases-generates a distinct reactivity state in astrocytes that alters the astrocytic secretome, leading to loss of synaptogenic function in vitro. Further, we establish that the same PERK-P-dependent astrocyte reactivity state is harmful to neurons in vivo in mice with prion neurodegeneration. Critically, targeting this signaling exclusively in astrocytes during prion disease is alone sufficient to prevent neuronal loss and significantly prolongs survival. Thus, the astrocyte reactivity state resulting from UPR over-activation is a distinct pathogenic mechanism that can by itself be effectively targeted for neuroprotection.

Topics: Animals; Astrocytes; eIF-2 Kinase; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Eukaryotic Initiation Factor-2B; In Vitro Techniques; Memory; Mice; Neurodegenerative Diseases; Phosphorylation; Prion Diseases; Protein Biosynthesis; Protein Phosphatase 1; Signal Transduction; Synapses; Thapsigargin; Transcriptome; Tunicamycin; Unfolded Protein Response

We have investigated the impact of endoplasmic reticulum (ER) stress, which is often implicated in neurodegenerative diseases, on the expression of Hrd1, an E3 ubiquitin ligase that plays a central role in the process of ER-associated degradation (ERAD).. SH-SY5Y neuroblastoma cells, a frequently used model for studying neurotoxicity in dopaminergic neurons and the mechanisms of neurodegeneration associated with Parkinson's disease, and parental SK-N-SH cells were studied.. We demonstrate that ER stress, induced by thapsigargin or tunicamycin, correlates with the increased expression of Hrd1 in both SH-SY5Y and SK-N-SH cells. Inhibition of PERK does not significantly suppress the thapsigargin- or tunicamycin-induced expression of Hrd1. Nevertheless, PERK inhibition has a positive effect on the survival of SH-SY5Y cells treated with thapsigargin but not on those treated with tunicamycin. Inhibition of IRE1 associated with the inhibition of XBP1 splicing does not affect the survival of SH-SY5Y cells treated with either thapsigargin or tunicamycin but results in the complete suppression of both the thapsigargin- and tunicamycin-induced expression of Hrd1.. Thus, the ER-stress-induced expression of Hrd1 in SH-SY5Y depends on Hrd1 transcription activation, which is a consequence of IRE1 but not of PERK activation.

Topics: Anti-Infective Agents; Cell Line, Tumor; eIF-2 Kinase; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Inhibitors; Humans; Neurodegenerative Diseases; Protein Serine-Threonine Kinases; Thapsigargin; Tunicamycin; Ubiquitin-Protein Ligases

The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons.. Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording.. Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM).. The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Topics: Calcium; Calcium-Transporting ATPases; Cell Differentiation; Cells, Cultured; Flavanones; Gene Expression; Humans; Induced Pluripotent Stem Cells; Lithium; Membrane Potentials; Neoplasm Proteins; Neurodegenerative Diseases; Neurons; NF-kappa B; ORAI1 Protein; Patch-Clamp Techniques; Stromal Interaction Molecule 1; Thapsigargin

Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases.

Topics: Acetylcysteine; Animals; Antibodies, Monoclonal; Brain; Cell Death; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; HeLa Cells; Humans; Male; Mice; Molecular Chaperones; Mutation, Missense; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Denaturation; Protein Structure, Tertiary; Thapsigargin; Ubiquitinated Proteins

Endoplasmic reticulum stress commonly causes neuronal damage in a lot of neurodegenerative diseases. In this study, we examined neuroprotective effect of bovine milk phospholipid fraction (mPL) on mouse neuroblastoma Neuro2a cells from endoplasmic reticulum (ER) stress induced cell death. Neuro2a cells were induced cell death by ER stressor tunicamycin (TM) or thapsigargin (TG), and studied whether mPL could attenuate the toxicity. By preincubation with mPL, the cell viabilities were significantly increased in TM or TG treated cells, and caspase-12 activated cells induced by TM or TG treatment were significantly decreased. Protein kinase C inhibitor GF109203x significantly reduced the protective effect on TM induced cell death, and autophagy inhibitor 3-methyladenine reduced the protective effect on TM or TG induced cell death. Moreover, preincubation with mPL significantly stimulated autophagosomes formation observed by dansylcadaverine staining. Our data suggest that mPL will be applicable to prevent neurodegenerative diseases caused by ER stress.

Topics: Animals; Autophagy; Caspase 12; Cell Death; Cell Line, Tumor; Endoplasmic Reticulum Stress; Mice; Milk; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Phospholipids; Protein Kinase C; Signal Transduction; Thapsigargin; Tunicamycin

Endoplasmic reticulum (ER) stress is involved in neurodegenerative diseases, and the KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR) plays a key role in ER quality control and in the ER stress response. The subcellular distribution of KDELR is dynamic and related to its ligand binding status and its expression level. Here, we show that KDELR mRNA is upregulated upon thapsigargin treatment, which induces ER stress. Moreover, overexpressed KDELR partially redistributes to the lysosome and activates autophagy. The R169N mutant, a ligand binding-defective form of KDELR, and D193N, a transport-defective form of KDELR, both fail to trigger autophagy. Overexpression of KDELR activates extracellular signal-regulated kinases (ERKs). Both the activation of ERKs and autophagy induced by KDELR could be blocked by PD98059, an inhibitor of mitogen extracellular kinase 1 (MEK1). The overexpression of some neurodegenerative disease-related proteins, such as amyotrophic lateral sclerosis (ALS)-linked G93A superoxide dismutase 1 (SOD1), Parkinson's disease-associated A53T alpha-synuclein and Huntington's disease-related expanded huntingtin, increase the mRNA levels of KDELR. Moreover, the overexpressed KDELR promotes the clearance of these disease proteins through autophagy. Taken together, our data provide evidence that KDELR, as a novel inducer of autophagy, participates in the degradation of misfolded neurodegenerative disease-related proteins.

Topics: Autophagy; Cells; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinases; Neurodegenerative Diseases; Receptors, Peptide; RNA, Small Interfering; Signal Transduction; Thapsigargin; Up-Regulation

To demonstrate that elevated pressure increases the peptidyl arginine deiminase 2 (PAD2) expression in cultured astrocytes in vitro that can be modulated by pharmacological agents modulating intracellular calcium.. Isolated rat brain astrocytes were subjected to pressure treatment. Western and immunohistochemical analyses detected PAD2 protein expression. Calcium measurements were achieved employing fluorescence-based microscopic imaging and quantification system. Experiments were repeated with human optic nerve head-derived astrocytes.. PAD2 has recently been shown to be associated with glaucomatous optic nerve. Astrocytes subjected to pressure (25-100 mmHg) show elevated level of PAD2, increased intracellular calcium, and concomitant citrullination but not significant cell death. PAD2 expression in response to elevated pressure may play a role in glaucomatous neurodegeneration. Pressure-treated astrocytes were also subjected to thapsigargin (50-250 nM) treatment, but it is unclear whether this had any further effect in increasing PAD2 expression. Conversely, treatment with calcium chelating agent BAPTA-AM (50-250 nM) results in decreased intracellular calcium concentration and PAD2.. These results suggest calcium modulation could be exploited as therapeutic strategy to modulate pressure-induced PAD2 expression and citrullination.

Topics: Animals; Astrocytes; Blotting, Western; Calcium; Cells, Cultured; Chelating Agents; Child; Egtazic Acid; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Humans; Hydrolases; Male; Microscopy, Fluorescence; Neurodegenerative Diseases; Osmotic Pressure; Protein-Arginine Deiminase Type 2; Protein-Arginine Deiminases; Rats; Rats, Sprague-Dawley; Thapsigargin

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Topics: Calcium; Calcium Signaling; Carbachol; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cytological Techniques; Cytosol; DNA; Electron Transport; Fluorescent Dyes; Homeostasis; Humans; Membrane Potentials; Microscopy, Electron; Mitochondria; Mitochondrial Myopathies; Neuroblastoma; Neurodegenerative Diseases; Thapsigargin; Tumor Cells, Cultured