Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes

Background

Hypoxia/reoxygenation(H/R)-induced apoptosis of cardiomyocytes plays an important role in myocardial injury. Lycopene is a potent antioxidant carotenoid that has been shown to have protective properties on cardiovascular system. The aim of the present study is to investigate the potential for lycopene to protect the cardiomyocytes exposed to H/R. Moreover, the effect on mitochondrial function upon lycopene exposure was assessed.

Methods and Findings

Primary cardiomyocytes were isolated from neonatal mouse and established an in vitro model of H/R which resembles ischemia/reperfusion in vivo. The pretreatment of cardiomyocytes with 5 µM lycopene significantly reduced the extent of apoptosis detected by TUNEL assays. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and the process of mitochondria-mediated apoptosis were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of the mitochondrial permeability transition pore (mPTP) by reducing the intracellular reactive oxygen species (ROS) levels and inhibiting the increase of malondialdehyde (MDA) levels caused by H/R. Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in lycopene-treated cultures.

Conclusion

The present results suggested that lycopene possesses great pharmacological potential in protecting against H/R-induced apoptosis. Importantly, the protective effects of lycopene may be attributed to its roles in improving mitochondrial function in H/R-treated cardiomyocytes.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

Overexpression of Extracellular Superoxide Dismutase Protects against Brain Injury Induced by Chronic Hypoxia

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of ¹⁸FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.     

Ciliary Neurotrophic Factor Protects Striatal Neurons against Excitotoxicity by Enhancing Glial Glutamate Uptake

Ciliary neurotrophic factor (CNTF) is a potent neuroprotective cytokine in different animal models of glutamate-induced excitotoxicity, although its action mechanisms are still poorly characterized. We tested the hypothesis that an increased function of glial glutamate transporters (GTs) could underlie CNTF-mediated neuroprotection. We show that neuronal loss induced by in vivo striatal injection of the excitotoxin quinolinic acid (QA) was significantly reduced (by ∼75%) in CNTF-treated animals. In striatal slices, acute QA application dramatically inhibited corticostriatal field potentials (FPs), whose recovery was significantly higher in CNTF rats compared to controls (∼40% vs. ∼7%), confirming an enhanced resistance to excitotoxicity. The GT inhibitor dl-threo-β-benzyloxyaspartate greatly reduced FP recovery in CNTF rats, supporting the role of GT in CNTF-mediated neuroprotection. Whole-cell patch-clamp recordings from striatal medium spiny neurons showed no alteration of basic properties of striatal glutamatergic transmission in CNTF animals, but the increased effect of a low-affinity competitive glutamate receptor antagonist (γ-d-glutamylglycine) also suggested an enhanced GT function. These data strongly support our hypothesis that CNTF is neuroprotective via an increased function of glial GTs, and further confirms the therapeutic potential of CNTF for the clinical treatment of progressive neurodegenerative diseases involving glutamate overflow.     

A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.     

Caspase-3 Protects Stressed Organs against Cell Death

The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.     

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4^-/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4^-/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4^-/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.     

溶血磷脂酸抗缺氧无血清诱导的大鼠骨髓间充质干细胞凋亡作用

在体外缺氧无血清条件下模拟心肌缺血微环境,研究溶血磷脂酸(LPA)对骨髓间充质干细胞(BMMSCs)的抗凋亡作用.应用Hoechst33342染色和膜联蛋白V/PI双染流式细胞术观察细胞凋亡,并利用Western印迹方法检测ERK1/2和Akt的磷酸化水平变化.结果表明缺氧无血清条件引起明显的细胞凋亡,LPA处理6 h和1 6 h 组细胞凋亡率较缺氧无血清组显著降低(P＜0.05),而1 h组细胞凋亡率与缺氧无血清组无显著性差异.LPA处理组(1 h,6h,1 6 h组)ERK1/2磷酸化水平较缺氧无血清组降低,Akt磷酸化水平较缺氧无血清组增高.在缺氧无血清环境中,LPA有利于增强BMMSCs的抗凋亡能力,提高BMMSCs存活率,为临床提高BMMSCs治疗缺血性心肌病的疗效提供理论依据.     

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca²⁺ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca²⁺ responses in GFP-tagged neurons, a non-green fluorescent Ca²⁺ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca²⁺ indicator fura-2 has been used in combination with GFP-tagged neurons¹. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals². Therefore, we decided to use a red fluorescent Ca²⁺ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons³. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons⁴. Fura-red based Ca²⁺ imaging was also successfully combined with GFP-tagged pancreatic β-cells and GFP-tagged receptors expressed in HEK cells^5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium⁷. Therefore, the fluorescence of resting neurons with low Ca²⁺ concentration has relatively high intensity. It should be noted, that other red Ca²⁺-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.     

Poloxamer 188 Protects Neurons against Ischemia/Reperfusion Injury through Preserving Integrity of Cell Membranes and Blood Brain Barrier

Poloxamer 188 (P188), a multiblock copolymer surfactant, has been shown to protect against ischemic tissue injury of cardiac muscle, testes and skeletal muscle, but the mechanisms have not been fully understood. In this study, we explored whether P188 had a protective effect against cerebral ischemia/reperfusion injury and its underlying mechanisms. The in vivo results showed that P188 significantly reduced the infarct volume, ameliorated the brain edema and neurological symptoms 24 h after ischemia/reperfusion. In the long-term outcome study, P188 markedly alleviated brain atrophy and motor impairments and increased survival rate in 3 weeks of post stroke period. Additionally, P188 protected cultured hippucampal HT22 cells against oxygen–glucose deprivation and reoxygenation (OGD/R) injury. The ability in membrane sealing was assessed with two fluorescent membrane-impermeant dyes. The results showed that P188 treatment significantly reduced the PI-positive cells following ischemia/reperfusion injury and repaired the HT22 cell membrane rupture induced by Triton X-100. In addition, P188 inhibited ischemia/reperfusion-induced activation of matrix metalloproteinase (MMP)-9 and leakage of Evans blue. Therefore, the present study concludes that P188 can protect against cerebral ischemia/reperfusion injury, and the protection involves multi-mechanisms in addition to the membrane resealing.     

Blocking Nuclear Factor-Kappa B Protects against Diet-Induced Hepatic Steatosis and Insulin Resistance in Mice

Inflammation critically contributes to the development of various metabolic diseases. However, the effects of inhibiting inflammatory signaling on hepatic steatosis and insulin resistance, as well as the underlying mechanisms remain obscure. In the current study, male C57BL/6J mice were fed a chow diet or high-fat diet (HFD) for 8 weeks. HFD-fed mice were respectively treated with p65 siRNA, non-silence control siRNA or vehicle every 4^th day for the last 4 weeks. Vehicle-treated (HF) and non-silence siRNA-treated (HFNS) mice displayed overt inflammation, hepatic steatosis and insulin resistance compared with chow-diet-fed (NC) mice. Upon treatment with NF-κB p65 siRNA, HFD-fed (HFPS) mice were protected from hepatic steatosis and insulin resistance. Furthermore, Atg7 and Beclin1 expressions and p-AMPK were increased while p-mTOR was decreased in livers of HFPS mice in relative to HF and HFNS mice. These results suggest a crosslink between NF-κB signaling pathway and liver AMPK/mTOR/autophagy axis in the context of hepatic steatosis and insulin resistance.     

Parkin Protects Dopaminergic Neurons against Microtubule-depolymerizing Toxins by Attenuating Microtubule-associated Protein Kinase Activation

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD). Our previous study has shown that parkin stabilizes microtubules through strong interactions mediated by three independent domains. We found that each of the three microtubule-binding domains of parkin was sufficient to reduce MAP kinase activation induced by microtubule depolymerization. The ability to attenuate microtubule depolymerization and the ensuing MAP kinase activation was abrogated in B-lymphocytes and fibroblasts derived from PD patients with parkin mutations such as exon 4 deletion. Such mutations produced truncated parkin proteins lacking any microtubule binding domain and prevented parkin from protecting midbrain dopaminergic neurons against microtubule-depolymerizing toxins such as rotenone or colchicine. Consistent with these, blocking MAP kinase activation in midbrain dopaminergic neurons by knocking down MAP kinase kinases (MKK) significantly reduced the selective toxicity of rotenone or colchicine. Conversely, overexpression of MAP kinases caused marked toxicities that were significantly attenuated by parkin. Thus, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins such as rotenone by stabilizing microtubules to attenuate MAP kinase activation.Mitogen-activated protein kinases are a superfamily of kinases that include the extracellular signal-related kinases (ERK1/2),² Jun N-terminal kinases (JNK1/2/3), and p38 proteins (p38α/β/γ/δ) in mammalian species (1). Initially, MAP kinase stood for microtubule-associated protein kinase because microtubule-associated proteins such as MAP2 are excellent substrates of MAP kinases (2, 3). Previous studies have shown that a significant portion of ERK is associated with microtubules (4). It has also been shown that JNK1 is required for the maintenance of microtubule integrity in neurons through controlling the phosphorylation states of MAP2 and MAP1B (5). Phosphorylation of tau, an axon-enriched MAP, by p38δ promotes microtubule assembly (6). All MAP kinases are proline-directed kinases, preferring serine or threonine residues followed by proline. The abundance of such sites on many microtubule-associated proteins suggests that MAP kinases play critical roles in regulating the phosphorylation states of MAPs; hence, the dynamic properties of microtubules.The selective loss of dopaminergic (DA) neurons in substantia nigra is the pathological hallmark of Parkinson disease and directly contributes to its locomotor symptoms. Nigral DA neurons project to striatal target areas with very long axons, which rely on microtubules to transport dopamine vesicles over long distances. Our previous studies have shown that these dopaminergic neurons are particularly vulnerable to microtubule-depolymerizing agents including rotenone (7), an environmental toxin that causes PD-like symptoms and pathologies in animal models (8). Microtubule depolymerization disrupts vesicular transport, which significantly elevates oxidative stress due to increased oxidation of cytosolic dopamine leaked from vesicles (7). On the other hand our previous studies have shown that parkin, a protein-ubiquitin E3 ligase linked to Parkinson disease, strongly binds to microtubules (9) through redundant, high affinity interactions mediated by three independent domains (10). In addition, parkin increases the ubiquitination and degradation of both α- and β-tubulin (9), whose complex folding reactions are prone to produce misfolded intermediates (11). These results suggest that parkin plays an important role in maintaining the stability and normal functions of microtubules, which are critically involved in the survival of nigral DA neurons (12).In the present study we examined the impact of parkin on MAP kinase activation induced by microtubule depolymerization. Our results showed that MAP kinases, including JNK, ERK, and p38, were activated by microtubule-depolymerizing agents such as colchicine and nocodazole. This effect was greatly attenuated by overexpression of wild-type parkin or any one of its three microtubule binding domains. The ability of parkin to suppress microtubule depolymerization and the ensuing MAP kinase activation was abrogated in PD patients with parkin mutations such as exon 4 deletion, which produced a truncated protein lacking any microtubule-binding domain. This mutation also prevented parkin from protecting dopaminergic neurons against the selective toxicity of microtubule-depolymerizing toxins such as rotenone or colchicine. Blocking MAP kinase activation by small interfering RNA (siRNA) of MAP kinase kinases significantly reduced the selective toxicity of rotenone or colchicine, whereas overexpression of MAP kinases produced toxicities that were significantly reduced by parkin. Together, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins by stabilizing microtubules to rein in MAP kinase activation.     

Reducing Hypothalamic Stem Cell Senescence Protects against Aging-Associated Physiological Decline

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Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin’s mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim^−/−) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim^−/−mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin’s enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim^−/−mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim^−/−mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim^−/−mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young’s moduli of normal and vim^−/−mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.     

Inhibiting PTEN Protects Hippocampal Neurons against Stretch Injury by Decreasing Membrane Translocation of AMPA Receptor GluR2 Subunit

The AMPA type of glutamate receptors (AMPARs)-mediated excitotoxicity is involved in the secondary neuronal death following traumatic brain injury (TBI). But the underlying cellular and molecular mechanisms remain unclear. In this study, the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GluR2-lacking AMPARs mediated neuronal death was investigated through an in vitro stretch injury model of neurons. It was indicated that both the mRNA and protein levels of PTEN were increased in cultured hippocampal neurons after stretch injury, which was associated with the decreasing expression of GluR2 subunits on the surface of neuronal membrane. Inhibition of PTEN activity by its inhibitor can promote the survival of neurons through preventing reduction of GluR2 on membrane. Moreover, the effect of inhibiting GluR2-lacking AMPARs was similar to PTEN suppression-mediated neuroprotective effect in stretch injury-induced neuronal death. Further evidence identified that the total GluR2 protein of neurons was not changed in all groups. So inhibition of PTEN or blockage of GluR2-lacking AMPARs may attenuate the death of hippocampal neurons post injury through decreasing the translocation of GluR2 subunit on the membrane effectively.     

Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy

The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm² in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm² in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.     

Bcl-2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin-Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca²⁺-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca²⁺ ([Ca²⁺]_i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca²⁺ diminished somewhat the size of the initial increase of [Ca²⁺]_i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La³⁺ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca²⁺ chelator, inhibited the peak [Ca²⁺]_i caused by thapsigargin but did not inhibit the sustained elevation of [Ca²⁺]_i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto-oncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca²⁺]_i or the elevation of [Ca²⁺]_i induced by thapsigargin. Our results suggest that abnormal Ca²⁺ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca²⁺ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca²⁺ release in GT1-7 neuronal cells.     

Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer''s disease, and other neurodegenerative disorders.