Neuronal Apoptosis In PC12 Cells Research Articles

IGFBP5 Promotes Neuronal Apoptosis in a 6-OHDA-Toxicant Model of Parkinson’s Disease by Inhibiting the Sonic Hedgehog Signaling Pathway

Introduction: Parkinson’s disease (PD) is the most common neurodegenerative disease worldwide. Studies have shown that insulin-like growth factor-binding protein 5 (IGFBP5) may contribute to methamphetamine-induced neurotoxicity and neuronal apoptosis in PC-12 cells and rat striatum. Here, we studied the expression and role of IGFBP5 in the 6-OHDA-toxicant model of PD. Methods: PC-12 and SH-SY5Y cells were exposed to 50 μm 6-OHDA for 24 h. qRT-PCR, western blotting, CCK-8 assay, EdU staining, annexin V staining, and immunofluorescence were performed to study the effects of IGFBP5-specific siRNAs. The effects of IGFBP5 on a rat 6-OHDA model of PD were confirmed by performing behavioral tests, tyrosine hydroxylase (TH) immunofluorescence staining, and western blotting. Results: In the GSE7621 dataset, IGFBP5 was highly expressed in the substantia nigra tissues of PD patients compared to healthy controls. In PC-12 and SH-SY5Y cells, IGFBP5 was upregulated following 6-OHDA exposure in a dose-dependent manner. Silencing of IGFBP5 promoted PC-12 and SH-SY5Y proliferation and inhibited apoptosis under 6-OHDA stimulation. Silencing of IGFBP5 relieved 6-OHDA-induced TH-positive neuron loss. Hedgehog signaling pathway was predicted as a downstream signaling pathway of IGFBP5. Negative regulation between IGFBP5 and sonic hedgehog (SHH) signaling pathway was confirmed in vitro. The effects of IGFBP5 silencing on SH-SY5Y cells were partially reversed using cyclopamine, a direct inhibitor of the SHH signaling pathway. In addition, silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. Conclusion: Elevated IGFBP5 expression may be involved in 6-OHDA-induced neurotoxicity through regulation of the SHH signaling pathway.

Dexmedetomidine attenuates oxygen-glucose deprivation/ reperfusion-induced inflammation through the miR-17-5p/ TLR4/ NF-\u03baB axis

BackgroundDexmedetomidine (DEX) is a selective agonist of α2-adrenergic receptors with anesthetic activity and neuroprotective benefits. However, its mechanism of action at the molecular level remains poorly defined. In this study, we investigated the protective effects of DEX on oxygen-glucose deprivation/ reperfusion (OGD/R)-induced neuronal apoptosis in PC12 cells, and evaluated its underlying mechanism(s) of neuroprotection and anti-inflammation.MethodsAn OGD/R model in PC12 cells was established. PC12 cells were cultured and divided into control, OGD/R, and OGD/R + DEX (1 μM, 10 μM, 50 μM) groups. Cell apoptosis was analyzed by flow cytometry and expression profiles were determined by qRT-PCR, western blot analysis, and enzyme linked immunosorbent assays (ELISA). The interaction between miRNA and its downstream targets was evaluated through luciferase reporter assays.ResultsDEX significantly decreased apoptosis rates and inhibited interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) release (P < 0.05). While expression of the pro-apoptotic proteins Bax and Caspase-3 was down-regulated, expression of Bcl-2 was upregulated in a dose-dependent manner (P < 0.05). Interestingly, miR-17-5p expression was down-regulated in the OGD/R group (compared to controls). Toll-like receptor 4 (TLR4), a key regulator of nuclear factor kappa-B (NF-κB) signaling, was identified as a novel target of miR-17-5p in PC12 cells. miR-17-5p expression was upregulated in the OGD/R + DEX group, suppressing TLR4 expression and reducing the secretion of proinflammatory cytokines.ConclusionDEX inhibits OGD/R-induced inflammation and apoptosis in PC12 cells by increasing miR-17-5p expression, downregulating TLR4, and inhibiting NF-κB signaling.

Upregulated Expression of SSTR3 is Involved in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats.

Somatostatin which is a multifunctional growth hormone inhibitory neuropeptide shows diverse physiological effects, such as neurotransmission, cell growth, apoptosis, and endocrine signaling as well as exerts inhibitory effects on hormonal products and other secretory proteins. SSTR3 is a member of superfamily of somatostatin receptors (SSTR), which are G-protein-coupled plasma membrane receptors. Previous studies proved that SSTR3 regulates antiproliferative signaling and apoptosis in several cells. Here, we explored a potential role of SSTR3 in the regulation of neuronal apoptosis in the course of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. We found SSTR3 was significantly upregulated surrounding the hematoma after ICH by Western blot and immunohistochemistry. Double immunofluorescence-manifested SSTR3 was strikingly increased in neurons, not astrocytes or microglia. Moreover, increasing SSTR3 level was found to be accompanied by the upregulation of p53, Bax, and active caspase-3 in vivo and in vitro studies. Furthermore, we detected that neuronal apoptosis marker active caspase-3 was co-localized with SSTR3 suggesting its potential role in neuronal apoptosis. In addition, in vitro study, revealed that SSTR3 knockdown specifically resulted in reducing neuronal apoptosis in PC12 cells, which further indicated that SSTR3 might exert its pro-apoptotic function on neuronal apoptosis. All our findings suggested that upregulated SSTR3 may be involved in neuronal apoptosis after ICH.

Protective Effect of Psoralea corylifolia L. Seed Extract against Palmitate-Induced Neuronal Apoptosis in PC12 Cells.

The extract of Psoralea corylifolia seeds (PCE) has been widely used as a herbal medicine because of its beneficial effect on human health. In this study, we investigated the protective effects and molecular mechanisms of PCE on palmitate- (PA-) induced toxicity in PC12 cells, a neuron-like cell line. PCE significantly increased cell viability in PA-treated PC12 cells and showed antiapoptotic effects, as evidenced by decreased expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, and bax protein as well as increased expression of bcl-2 protein. In addition, PCE treatment reduced PA-induced reactive oxygen species production and upregulated mRNA levels of antioxidant genes such as nuclear factor (erythroid-derived 2)-like 2 and heme oxygenase 1. Moreover, PCE treatment recovered the expression of autophagy marker genes such as beclin-1 and p62, which was decreased by PA treatment. Treatment with isopsoralen, one of the major components of PCE extract, also recovered the expression of autophagy marker genes and reduced PA-induced apoptosis. In conclusion, PCE exerts protective effects against lipotoxicity via its antioxidant function, and this effect is mediated by activation of autophagy. PCE might be a potential pharmacological agent to protect against neuronal cell injury caused by oxidative stress or lipotoxicity.

Upregulated expression of SSTR1 is involved in neuronal apoptosis and is coupled to the reduction of bcl-2 following intracerebral hemorrhage in adult rats.

Somatostatins are peptide hormones that regulate diverse cellular processes, such as neurotransmission, cell proliferation, apoptosis, and endocrine signaling as well as inhibiting the release of many hormones and other secretory proteins. SSTR1 is a member of the superfamily of somatostatin receptors possessing seven-transmembrane segments. Aberrant expression of SSTR1 has been implicated in several human diseases, including pseudotumor cerebri, and oncogenic osteomalacia. In this study, we investigated a potential role of SSTR1 in the regulation of neuronal apoptosis in the course of intracerebral hemorrhage (ICH). A rat ICH model in the caudate putamen was established and subjected to behavioral tests. Western blot and immunohistochemistry indicated a remarkable up-regulation of SSTR1 expression surrounding the hematoma after ICH. Double-labeled immunofluorescence showed that SSTR1 was mostly co-localized with neurons, and was rarely distributed in activated astrocytes and microglia. Additionally, SSTR1 co-localized with active-caspase-3 and bcl-2 around the hematoma. The expression of active-caspase-3 was parallel with that of SSTR1 in a time-dependent manner. In addition, SSTR1 knockdown specifically resulted in reduced neuronal apoptosis in PC12 cells. All our findings suggested that up-regulated SSTR1 contributed to neuronal apoptosis after ICH, which was accompanied with reduced expression of bcl-2.

Protective effects of Gynostemma pentaphyllum polysaccharides on PC12 cells impaired by MPP+

The aim of the present study was to explore the neuroprotective effects of Gynostemma pentaphyllum polysaccharides (GP) in a 1-methyl-4-phenylpyridiniumion (MPP+)-induced cellular model of Parkinson's disease (PD) and the underlying mechanisms. Our results indicated that exposure of PC12 cells to 1mM MPP+ significantly decreased the cell viability when examined by MTT assay, LDH assay, and annexin-V-FITC and propidium iodide (PI) apoptosis detection assays. MPP+-induced apoptosis in PC12 cells was accompanied by an increased Bax/Bcl-2 ratio, release of mitochondrial cytochrome c into the cytosol, activation of caspase-3/9 and cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment of PC12 cells with 50μg/ml GP prior to MPP+ exposure effectively attenuated the cytotoxicity and improved cell viability via inhibiting elevated Bax/Bcl-2 ratio, as well as the release of cytosolic cytochrome c. Furthermore, GP was effective in attenuating caspase-3/9 activation and cleavage of PARP in MPP+-exposed PC12 cells. These results suggest that the GP has protective effects against MPP+-induced neuronal apoptosis in PC12 cells by suppressing apoptosis-related protein, and therefore, might likely be a promising candidate for the treatment of Parkinson's disease (PD).

Effect of isoflurane preconditioning on glutamate-induced apoptosis in rat neuronal PC12 cells

Objective To investigate the effect of isoflurane preconditioning on glutamate-induced apoptosis in rat neuronal PC12 cells.Methods The PC12 cells were cultured for 5 d with nerve growth factor in vitro.The cells were seeded into 6-cm-diameter culture dishes (3 ml/dish) or 6-well plates (2 ml/well) with the density of 5 × 104/ml and randomly divided into 4 groups (n =18 each): normal control group (group C); glutamate group (group G) ;glutamate + isoflurane group (group GI) and glutamate + isoflurane + xestospongin C (an antngon of inositol trisphosphate receptors) group (group GIX).The neuronal PC12 cells were exposed to glutamate 500 μmol/L in groups G,GI and GIX.The neuronal PC12 cells were exposed to 1.2％ isoflurane for 2 h in groups GI and GIX.Xestospongin C was added to the culture medium immediately before isoflurane preconditioning.Glutamate was added to the culture medium at 10 min after isoflurane preconditioning in groups GI and GIX.The cells were collected from six dishes or wells in each group after being incubated with glutamate for 20 min.The apoptosis and mitochondiral membrane potential (MMP) were assessed by flow cytometry.Intracellular Ca2+ concentration ([ Ca2+ ] i)was detected by confocal fluorescence microscopy.Results Compared with group C,the apoptotic rate and [Ca2+ ]i were significantly increased and MMP was decreased in groups G and GIX ( P ＜ 0.01 ),but there was no significant difference in the variables mentioned above in group GI (P ＞ 0.05).Compared with group G,the apoptotic rate and [ Ca2 + ]i were significantly decreased and MMP was increased in groups GI and GIX ( P ＜ 0.05 or 0.01).Compared with group GI,the apoptotic rate and [Ca2+ ]i were significantly increased and MMP was decreased in group GIX ( P ＜ 0.01 ).Conclusion Isollurane preconditioning can inhibit apoptosis in rat neuronal PC12 cells by activating inositol trisphosphate receptors,inhibiting Ca2+ release from the endoplasmic reticulum and increasing MMP. Key words: Isoflurane; Ischemic preconditioing; Glutamic acid; PC12 cells; Neurons; Apoptosis

Studying the effect of LPS on cytotoxicity and apoptosis in PC12 neuronal cells: Role of Bax, Bcl-2, and Caspase-3 protein expression

Gram-negative bacteria and their endotoxins may be a causal or complicating factor in many serious diseases. Bacterial lipopolysaccharides (LPS) could potentially be human pathogens. Among the many disorders induced by LPS, neurodegenerative diseases such as Parkinson’s are reported and are of great interest. Despite the evidence on LPS-induced neurodegeneration, the exact mechanism is unknown. The purpose of this study was to investigate the cytotoxic effect of LPS and also to examine the involvement of Bax, pro-apoptotic, Bcl-2, anti-apoptotic, and caspase-3, the executioner of apoptosis, protein expression, during LPS-induced apoptosis in neuronal PC12 cells. The cell viability was evaluated by MTT assy. The expression of pro-apoptotic Bax and anti-apoptotic Bcl2 and caspase -3 protein expressions were measured by immunoblotting. The results showed that LPS could reduce cell viability after 72 h in a dose-dependent manner which was statistically significant in concentration of 200 µg/ml. In western blotting analysis, LPS (200 µg/ml) also enhanced expression of pro-apoptotic Bax and pro-caspase-3 proteins compared to controls, while the expression of Bcl-2 protein was not changed significantly. The Bax/Bcl-2 ratio was significantly increased in LPS-treated cells compared to controls. From the present results, it might be concluded that LPS can cause PC12 cell death, in which apoptosis plays an important role, possibly by the mitochondrial pathway through higher expression of the Bax as well as caspase 3 protein.

Neuroprotective effect of methanol extract of Phellodendri Cortex against 1‐methyl‐4‐phenylpyridinium (MPP+)‐induced apoptosis in PC‐12 cells

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP(+))-induced neuronal apoptosis in PC-12 cells have been investigated. MPP(+)-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP(+)-induced neuronal apoptosis in PC-12 cells.

Nerve Growth Factor Potentiates p53 DNA Binding but Inhibits Nitric Oxide-Induced Apoptosis in Neuronal PC12 Cells

NGF is recognized for its role in neuronal differentiation and maintenance. Differentiation of PC12 cells by NGF involves p53, a transcription factor that controls growth arrest and apoptosis. We investigated NGF influence over p53 activity during NO-induced apoptosis by sodium nitroprusside in differentiated and mitotic PC12 cells. NGF-differentiation produced increased p53 levels, nuclear localization and sequence-specific DNA binding. Apoptosis in mitotic cells also produced these events but the accompanying activation of caspases 1-10 and mitochondrial depolarization were inhibited during NGF differentiation and could be reversed in p53-silenced cells. Transcriptional regulation of PUMA and survivin expression were not inhibited by NGF, although NO-induced mitochondrial depolarization was dependent upon de novo gene transcription and only occurred in mitotic cells. We conclude that NGF mediates prosurvival signaling by increasing factors such as Bcl-2 and p21(Waf1/Cip1) without altering p53 transcriptional activity to inhibit mitochondrial depolarization, caspase activation and apoptosis.

Effects of mild and moderate hypothermia on apoptosis in neuronal PC12 cells

There is still a possibility that mild hypothermic therapy may be useful as a neuroprotective tool during the intraoperative period, although the mechanism of cerebral protection by mild hypothermia is not well understood. We hypothesized that mild hypothermia may be protective against cerebral ischaemia by inhibiting post-ischaemia apoptosis. In this study, we used serum-deprived PC12 cells as the neuronal apoptotic model and examined the direct effects of mild and moderate hypothermia. Apoptosis was induced by depriving the cell culture medium of serum, which is one of the most representative methods to induce apoptosis, but not necrosis, in PC12 cells. Effects of mild (35 and 33 degrees C) and moderate (31 and 29 degrees C) hypothermia on apoptosis were evaluated. Cytotoxicity (lactate dehydrogenase leakage) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. As a control, cells without induction of apoptosis were incubated under the same conditions as the apoptosis group. Without induction at 37 degrees C, cytotoxicity and the percentage of apoptotic cells were over 60 and 90%, respectively. At each temperature examined below 35 degrees C, significant decreases in cytotoxicity and the percentage of apoptotic cells were observed. Mean cytotoxicity at 31 and 29 degrees C was 50.2 (SD 4.2)% and 47.9 (4.4)%, respectively. The percentage of apoptotic cells at 31 and 29 degrees C was 42.5 (7.4)% and 36.5 (7.3)%, respectively. In the control group, cytotoxicity and the percentage of apoptotic cells were significantly higher at 29 degrees C than at 37 degrees C. Mild and moderate hypothermia (29-35 degrees C) inhibited apoptosis, although hypothermia below 30 degrees C may induce apoptosis in intact cells.

Enhancement of oxidative stress-induced apoptosis by Hsp105alpha in mouse embryonal F9 cells.

Hsp105alpha is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105alpha prevents stress-induced apoptosis in neuronal PC12 cells, and is a novel anti-apoptotic neuroprotective factor in the mammalian brain. On the other hand, we have also demonstrated that Hsp105alpha is expressed transiently at high levels during mouse embryogenesis and is found not only in various tissues but also in apoptotic cells. In the present study, to elucidate the role of Hsp105alpha during mouse embryogenesis, we established mouse embryonal F9 cell lines that constitutively over-express Hsp105alpha. Over-expression of Hsp105alpha enhanced hydrogen peroxide-induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerase cleavage, cytochrome c release and activation of p38 mitogen-activated protein kinase (p38). Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105alpha enhances activation of p38, release of cytochrome c and caspase activation. Hsp105alpha may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis.

Role of hsp105 in Protection against Stress-Induced Apoptosis in Neuronal PC12 Cells

hsp105α is a stress protein characteristically highly expressed in the brain compared with other tissues in mammals. Here, to examine whether hsp105α plays a pivotal role in the nervous system, we tested the capability of hsp105α to protect against apoptosis in rat neuronal PC12 cells. Various stress treatments such as serum deprivation, heat shock, hydrogen peroxide, etoposide, and actinomycin D induced apoptosis in PC12 cells with characteristic shrinking of nuclei and chromatin. However, PC12 cells that constitutively overexpressed mouse hsp105α exhibited a strong protective effect against apoptosis induced by these stress treatments. Cleavage of poly(ADP-ribose) polymerase induced in PC12 cells by these treatments was inhibited in the constitutively overexpressed hsp105α cells. Furthermore, c-Jun N-terminal kinase (JNK) was activated in the cells treated with heat shock but not other treatments, and the heat-induced JNK activation was inhibited by the constitutive expression of hsp105α.Thus, hsp105α prevents not only heat-induced apoptosis by inhibiting JNK activation, but also prevents the apoptosis induced by other stressors through different pathways, and may play important roles in neuronal protection.

Nitric oxide as a bifunctional regulator of apoptosis.

It was inevitable that important relationships between two of the most intensely studied topics in biomedical research, apoptosis and nitric oxide (NO), would become apparent. Apoptosis is essential to normal development as well as physiological cell turnover. Although apoptosis in excess can manifest as tissue damage, a failure to undergo apoptosis constitutes pathological cellular overgrowth. It is now evident that NO and its reaction products can either promote or prevent apoptosis in a multitude of settings. The ubiquitous distribution of the NO synthases and the remarkable diffusibility and diverse chemical reactivity of NO in biological systems make this molecule unique among the regulators of apoptosis. Understanding the factors that govern the consequences of NO exposure on cell viability and identifying the conditions in which NO regulation of apoptosis contribute to pathology are topics of considerable interest and potential importance. In this article, we will review the recent observations on NO as a regulator of apoptosis. Apoptosis, or programmed cell death, is distinguished from lytic or necrotic cell death by specific biochemical and structural events (see recent review in Reference 11 ). Apoptogenic signals trigger cell-specific signaling pathways, including protease activation, followed by the appearance of morphological changes characteristic of cells undergoing apoptosis, including condensation of nuclei and cytoplasm, blebbing of the cytoplasmic membranes, and finally fragmentation into apoptotic bodies that are phagocytosed by neighboring cells. The elucidation of the signaling events in apoptosis is occurring at a rapid pace and includes the identification of the key roles of cysteine proteases (known as caspases), Bcl-2 family members, and mitochondria. Caspases, the mammalian counterpart of ced-3 in Caenorhabditis elegans , are a family of cysteine proteases now known to contain at least 14 homologs. Ectopic expression of any of the caspase family proteases can cause apoptosis; however, not all caspase family …

Nitric oxide (NO.) and the nitrosonium cation (NO+) reduce mitochondrial membrane potential and trigger apoptosis in neuronal PC12 cells.

Nitric oxide and its redox related species exhibit both neuroprotective and neurotoxic effects. It has been suggested that the nitrosonium cation (NO') is neuroprotective towards NMDA toxicity, while the free radical nitric oxide (NO') is itself neurotoxic [I]. However, NO' releasers such as S-nitroso-N-acetyl penicillamine (SNAP) have also been shown to suppress the apoptotic death of neuronal PC12 cells following nerve growth factor (NGF) withdrawal [2]. In the present study we have investigated in detail the role of the two redox related species, NO' and NO', on neuronal apoptosis. We show that NO' and NO' will either prevent or trigger the apoptotic death of neuronal PC12 cells depending on the redox state of the cell, the concentration of donor species for NO' and NO' and the presence or absence of NGF in the culture medium. Neuronal PC 12 cells undergo apoptotic cell death following NGF withdrawal [3]. We first investigated the effects of low concentrations of NO' and NO' on the survival of PC12 cells

Induction of nitric oxide synthase and nitric oxide-mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor-alpha/lipopolysaccharide.

Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differentiated PC12 cells was associated with cell death characterized by features of apoptosis. The NOS inhibitors N-monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl prevented TNF-alpha/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-alpha/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of L-arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-alpha/LPS-induced loss of viability.