EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell–cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3^−/−), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3^−/− mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3^−/− mice, but not EphB3^−/− mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.     

Minocycline attenuates neuronal apoptosis and improves motor function after traumatic brain injury in rats

Minocycline is a type of tetracycline antibiotic with broad-spectrum antibacterial activity that has been demonstrated to protect the brain against a series of central nervous system diseases. However, the precise mechanisms of these neuroprotective actions remain unknown. In the present study, we found that minocycline treatment significantly reduced HT22 cell apoptosis in a mechanical cell injury model. In addition, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining confirmed the neuroprotective effects of minocycline in vivo through the inhibition of apoptosis in a rat model of controlled cortical impact (CCI) brain injury. The western blotting analysis revealed that minocycline treatment significantly downregulated the pro-apoptotic proteins BAX and cleaved caspase-3 and upregulated the anti-apoptotic protein BCL-2. Furthermore, the beam-walking test showed that the administration of minocycline ameliorated traumatic brain injury (TBI)-induced deficits in motor function. Taken together, these findings suggested that minocycline attenuated neuronal apoptosis and improved motor function following TBI.     

Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.     

Brain-derived respiring mitochondria exhibit homogeneous, complete and cyclosporin-sensitive permeability transition

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.     

颅脑创伤后大鼠脑组织脑红蛋白表达变化及其与神经元凋亡的关系研究

目的：研究大鼠弥漫性颅脑创伤后脑组织中脑红蛋白的表达变化情况,探究创伤后脑红蛋白表达变化及其与神经元凋亡的关系。方法：采用雄性SD大鼠50只,随机分为10组（n=5）空白对照组、伤后30min、1h、2h、6h、12h、24h、48h、72h和5d组。以Marmarou’s自由落体打击装置复制颅脑创伤模型,采用免疫组化技术检测伤后不同时间脑组织中脑红蛋白的表达情况及神经元凋亡相关基因Bax、Bcl-2表达情况,并对所得数据进行统计学分析。结果：致伤区皮层神经元脑红蛋白表达分别于伤后2h、72h呈现出两次高峰表达;伤后30min～1h、48～72h期间大脑皮层区脑红蛋白表达的上调均伴随着Bax/Bcl-2比值上升趋势减缓甚至呈现下降趋势。结论：弥漫性颅脑创伤后脑组织中脑红蛋白的高表达在一定程度上可以拮抗创伤应激及伤后继发缺血、缺氧性损伤所导致的神经元凋亡,在颅脑创伤的超早期（〈3h）、急性期（〈72h）可能具有一定的神经保护作用。     

Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11-deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.     

Finite element modelling of equestrian helmet impacts exposes the need to address rotational kinematics in future helmet designs

Jockey head injuries, especially concussions, are common in horse racing. Current helmets do help to reduce the severity and incidences of head injury, but the high concussion incidence rates suggest that there may be scope to improve the performance of equestrian helmets. Finite element simulations in ABAQUS/Explicit were used to model a realistic helmet model during standard helmeted rigid headform impacts and helmeted head model University College Dublin Brain Trauma Model (UCDBTM) impacts.

Current helmet standards for impact determine helmet performance based solely on linear acceleration. Brain injury-related values (stress and strain) from the UCDBTM showed that a performance improvement based on linear acceleration does not imply the same improvement in head injury-related brain tissue loads. It is recommended that angular kinematics be considered in future equestrian helmet standards, as angular acceleration was seen to correlate with stress and strain in the brain.     

配对螺旋样纤维(PHF)-Tau与神经细胞的死亡

神经纤维缠结(neurofibrillary tangles, NFT)是老年性痴呆病的重要病理特征,人类神经tau分子聚集形成的配对螺旋样纤维(paired helical filaments, PHF)是NFT的主要成分.最近研究表明,PHF与神经细胞的坏死和程序化死亡密切相关,其作用机理可能是诱发细胞内氧化自由基系统的启动而导致神经细胞死亡的.     

Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2⁺ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1⁺ microglial cells were proliferating more. A smaller, but significant number of GFAP⁺ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX⁺ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP⁺ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.     

The overexpression of RBM3 alleviates TBI‐induced behaviour impairment and AD‐like tauopathy in mice

The therapeutic hypothermia is an effective tool for TBI‐associated brain impairment, but its side effects limit in clinical routine use. Hypothermia up‐regulates RNA‐binding motif protein 3 (RBM3), which is verified to protect synaptic plasticity. Here, we found that cognitive and LTP deficits, loss of spines, AD‐like tau pathologies are displayed one month after TBI in mice. In contrast, the deficits of LTP and cognitive, loss of spines and tau abnormal phosphorylation at several sites are obviously reversed in TBI mice combined with hypothermia pre‐treatment (HT). But, the neuroprotective role of HT disappears in TBI mouse models under condition of blocking RBM3 expression with RBM3 shRNA. In other hand, overexpressing RBM3 by AAV‐RBM3 plasmid can mimic HT‐like neuroprotection against TBI‐induced chronic brain injuries, such as improving LTP and cognitive, loss of spines and tau hyperphosphorylation in TBI mouse models. Taken together, hypothermia pre‐treatment reverses TBI‐induced chronic AD‐like pathology and behaviour deficits in RBM3 expression dependent manner, RBM3 may be a potential target for neurodegeneration diseases including Alzheimer disease.     

Neuroprotective roles of pituitary adenylate cyclase-activating polypeptide in neurodegenerative diseases

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic bioactive peptide that was first isolated from an ovine hypothalamus in 1989. PACAP belongs to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily. PACAP is widely distributed in the central and peripheral nervous systems and acts as a neurotransmitter, neuromodulator, and neurotrophic factor via three major receptors (PAC1, VPAC1, and VPAC2). Recent studies have shown a neuroprotective role of PACAP using in vitro and in vivo models. In this review, we briefly summarize the current findings on the neurotrophic and neuroprotective effects of PACAP in different brain injury models, such as cerebral ischemia, Parkinson’s disease (PD), and Alzheimer’s disease (AD). This review will provide information for the future development of therapeutic strategies in treatment of these neurodegenerative diseases. [BMB Reports 2014; 47(7): 369-375]     

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini-gene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing.     

Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of alpha II-spectrin and alpha II-SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.     

创伤性脑损伤对大鼠海马骨架蛋白及学习记忆功能影响

创伤性脑损伤（traumatic brain injury,TBI）是极为常见的外伤性疾病,致死率和致残率很高。存活者伴随的空间认知功能障碍,给患者家庭和社会造成了极大的负担。目前,对TBI造成的空间记忆障碍缺乏系统研究。脑损伤后海马组织与记忆有关的分子以及组成神经元骨架的分子如何变化研究甚少。本研究采用Wistar大鼠为研究对象,并随机将其分为假手术（sham）组和创伤性脑损伤（TBI）组。TBI组再按致伤后时间长短分为6 h、12 h、24 h、72 h、15 d五个亚组。TBI组应用PinPointTM颅脑撞击器撞击而致伤,sham组不撞击。采用Morris水迷宫评价实验动物空间记忆能力;干湿重法测定脑含水量,评估脑水肿与海马水通道蛋白4（aquaporin-4,AQP-4）的相关性;海马神经元特异性核蛋白（neuron specific nuclear protein,NeuN）标记和免疫荧光检测评估TBI致大鼠神经元丢失情况;通过Western印迹检测TBI致海马骨架相关蛋白质和记忆相关蛋白质含量变化。本研究证实,与sham组相比,TBI组大鼠潜伏期明显增加［（61.98±12.82) s vs.(28.32±8.52) s,n=5,P<0.01,day 15］,探索时间明显缩短［（36.98±0.37) s vs. (73.68±5.09) s,n=5,P<0.01,day15］,表明脑创伤损害了动物的空间参考记忆能力和空间工作记忆能力。与sham组相比,TBI组大鼠海马AQP-4在蛋白质水平上的表达和脑含水量持续升高,15 d恢复正常;在12 h［（3.78±0.74),(83.78±0.35)%］和72 h［（3.49±0.85),(82.28±0.63)%］均形成两个波峰,n=5,P均<0.01,表明继发性脑损伤与持续脑水肿和海马AQP-4在蛋白质上的高表达有关。与sham组相比,NeuN标记和免疫荧光检测发现,TBI后24 h 致大鼠海马神经元丢失严重［（198.2±8.002) vs.(297.2±6.866) cells/mm2, n=5,P<0.01］,表明TBI动物的海马功能受损。与sham相比,TBI组海马神经元树突标志物微管结合蛋白2（microtubule associated proein 2,MAP2）和突触前终末特异性标记物突触素（synaptophysin,SYN）在蛋白质水平均伤后逐步降低（n=5,P均<0.01）,72 h［（0.55±0.05) vs.(1.27±0.08), (0.52±0.14) vs.(1.06±0.16), n=5,P均<0.01］降低最明显;TBI组形成神经元纤维缠结主要成分的过度磷酸化tau（ser404）,伤后逐步升高,72 h［（1.25±0.11)vs. (0.33±0.07), n=5,P<0.01］升高最明显。 MAP2、SYN和过度磷酸化的tau（ser404）检测指标的改变,表明脑损伤致神经元受损,神经元生长和损伤修复能力减弱,最终导致神经元骨架破环,TBI损害了动物的海马空间记忆能力。与sham组相比,TBI组大鼠海马环磷酸腺苷反应元件结合蛋白（cAMP response element binding protein,CREB）和磷酸化CREB ser133(phosphorylated CREB Ser133, pCREB Ser133)含量降低明显（n=5,P均<0.05）,表明脑损伤动物海马的存储记忆能力减弱;TBI组大鼠海马一般调控阻遏蛋白激酶2(general control nonderepressible 2 kinase,GCN2)蛋白质升高明显（n=5,P均<0.05）,表明脑损伤动物海马将新信息转化成长期记忆能力下降。本研究提示,创伤性脑损伤可使大鼠海马神经元骨架破坏,进而导致在学习记忆过程中起重要作用的分子蛋白质下调,抑制记忆储存的蛋白质（GCN2）上调,促使学习记忆功能障碍。     

创伤性脑损伤对大鼠海马骨架蛋白及学习记忆功能影响

创伤性脑损伤（traumatic brain injury,TBI）是极为常见的外伤性疾病,致死率和致残率很高。存活者伴随的空间认知功能障碍,给患者家庭和社会造成了极大的负担。目前,对TBI造成的空间记忆障碍缺乏系统研究。脑损伤后海马组织与记忆有关的分子以及组成神经元骨架的分子如何变化研究甚少。本研究采用Wistar大鼠为研究对象,并随机将其分为假手术（sham）组和创伤性脑损伤（TBI）组。TBI组再按致伤后时间长短分为6 h、12 h、24 h、72 h、15 d五个亚组。TBI组应用PinPointTM颅脑撞击器撞击而致伤,sham组不撞击。采用Morris水迷宫评价实验动物空间记忆能力;干湿重法测定脑含水量,评估脑水肿与海马水通道蛋白4（aquaporin-4,AQP-4）的相关性;海马神经元特异性核蛋白（neuron specific nuclear protein,NeuN）标记和免疫荧光检测评估TBI致大鼠神经元丢失情况;通过Western印迹检测TBI致海马骨架相关蛋白质和记忆相关蛋白质含量变化。本研究证实,与sham组相比,TBI组大鼠潜伏期明显增加［（61.98±12.82) s vs.(28.32±8.52) s,n=5,P<0.01,day 15］,探索时间明显缩短［（36.98±0.37) s vs. (73.68±5.09) s,n=5,P<0.01,day15］,表明脑创伤损害了动物的空间参考记忆能力和空间工作记忆能力。与sham组相比,TBI组大鼠海马AQP-4在蛋白质水平上的表达和脑含水量持续升高,15 d恢复正常;在12 h［（3.78±0.74),(83.78±0.35)%］和72 h［（3.49±0.85),(82.28±0.63)%］均形成两个波峰,n=5,P均<0.01,表明继发性脑损伤与持续脑水肿和海马AQP-4在蛋白质上的高表达有关。与sham组相比,NeuN标记和免疫荧光检测发现,TBI后24 h 致大鼠海马神经元丢失严重［（198.2±8.002) vs.(297.2±6.866) cells/mm2, n=5,P<0.01］,表明TBI动物的海马功能受损。与sham相比,TBI组海马神经元树突标志物微管结合蛋白2（microtubule associated proein 2,MAP2）和突触前终末特异性标记物突触素（synaptophysin,SYN）在蛋白质水平均伤后逐步降低（n=5,P均<0.01）,72 h［（0.55±0.05) vs.(1.27±0.08), (0.52±0.14) vs.(1.06±0.16), n=5,P均<0.01］降低最明显;TBI组形成神经元纤维缠结主要成分的过度磷酸化tau（ser404）,伤后逐步升高,72 h［（1.25±0.11)vs. (0.33±0.07), n=5,P<0.01］升高最明显。 MAP2、SYN和过度磷酸化的tau（ser404）检测指标的改变,表明脑损伤致神经元受损,神经元生长和损伤修复能力减弱,最终导致神经元骨架破环,TBI损害了动物的海马空间记忆能力。与sham组相比,TBI组大鼠海马环磷酸腺苷反应元件结合蛋白（cAMP response element binding protein,CREB）和磷酸化CREB ser133(phosphorylated CREB Ser133, pCREB Ser133)含量降低明显（n=5,P均<0.05）,表明脑损伤动物海马的存储记忆能力减弱;TBI组大鼠海马一般调控阻遏蛋白激酶2(general control nonderepressible 2 kinase,GCN2)蛋白质升高明显（n=5,P均<0.05）,表明脑损伤动物海马将新信息转化成长期记忆能力下降。本研究提示,创伤性脑损伤可使大鼠海马神经元骨架破坏,进而导致在学习记忆过程中起重要作用的分子蛋白质下调,抑制记忆储存的蛋白质（GCN2）上调,促使学习记忆功能障碍。     

蝙蝠葛碱拮抗缓激肽引起的钙稳态改变 及细胞骨架蛋白的异常磷酸

为研究蝙蝠葛碱 (dauricine , Dau) 拮抗缓激肽 (bradykinin , BK) 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用,采用双波长荧光分光光度计测定细胞内钙离子浓度 ([Ca2+] i) ,用 MTT 法检测细胞代谢水平,用免疫组织化学方法观察 tau 蛋白表达和磷酸化 . 结果表明,Dau (3 μmol/L , 6 μmol/L) 可抑制 BK 诱导的 [Ca2+]i 升高,保护 BK 引起的神经元代谢降低,拮抗 BK 引起的 tau 蛋白异常磷酸化和聚集 . 结果提示： Dau 可拮抗 BK 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用 .     

FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death

Two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) and emission spectral imaging (ESI) are powerful tools for fluorescence resonance energy transfer (FRET) measurement. In this study, we use these two techniques to analyze caspase-3 activation inside single living cells during anticancer drug-induced human lung adenocarcinoma (ASTC-a-1) cell death. TPE-ESI of SCAT3, a caspase-3 indicator based on FRET, was performed inside single living cell stably expressing SCAT3. The TPE-ESI measurement was performed using 780 nm excitation which was considered to selectively excite the donor ECFP of SCAT3 by measuring the emission ratio of 526 to 476 nm. The emission peak at 526 nm disappeared and that of 476 nm increased after STS or bufalin treatment, but taxol treatment did not induce a significant change for the SCAT3 emission spectra, indicating that caspase-3 was activated during STS- or bufalin-induced cell apoptosis, but was not involved in taxol-induced PCD. Fluorescence lifetime of ECFP inside living cells was acquired using FLIM. The lifetime of ECFP was the same as that of the control group after taxol treatment, but increased from 1.83 ± 0.02 to 2.05 ± 0.03 and 1.90 ± 0.03 ns, respectively after STS and bufalin treatment, which agree with the results obtained using TPE-ESI. Taken together, TPE-FLIM and ESI analysis were proved to be valuable approaches for monitoring caspase-3 activation inside single living cells. W. Pan and J. Qu contributed equally to this study.     

Short interfering RNA against the PDCD5 attenuates cell apoptosis and caspase-3 activity induced by Bax overexpression

The programmed cell death 5 (PDCD5) protein plays an important apoptosis-accelerating role in cells undergoing apoptosis. Decreased expression of PDCD5 has been detected in various human carcinomas. Here we describe that one potent short interfering RNA (siRNA) against the PDCD5 (siPDCD5) specifically inhibits the expression of PDCD5 at both the mRNA and protein level. Cells with decreased PDCD5 expression displayed reduced sensitivity to an apoptotic stimulus induced by Bax overexpression in HeLa, HEK293 and 293T cell lines. Furthermore, we also show that siPDCD5 inhibited both caspase-3 activity and procaspase-3 cleavage. Suppressed expression of PDCD5 attenuates the release of cytochrome c from mitochondria to cytosol induced by Bax overexpression. This phenomenon is accompanied by the reduced translocation of Bax from the cytosol to mitochondria. MTT assay shows that targeted suppression of PDCD5 expression markedly promoted cell proliferation in Hela and HEK293 cell lines. Our data suggests that PDCD5 may exert its effects through pathway of mitochondria by modulating Bax translocation, cytochrome c release and caspase 3 activation directly or indirectly, and that decreased PDCD5 expression may be one of the mechanisms by which tumor cells achieve resistance to apoptotic stimulus induced by anticancer drugs.