世界生命科学前沿动态周报（五十九）

（9.26-10.2/2011）
美宝国际集团:陶国新 

主要内容：用诱导多能干细胞纠正镰刀状细胞疾病的基因突变；活化蛋白激酶乙酰化控制酵母内源性衰老；肠促胰岛素增强胰岛生命力；神经细胞生存的关键；人体脂肪细胞脂质翻转造成代谢疾病；人脑神经细胞迁移通道及其在婴儿期的衰退。

焦点动态：用诱导多能干细胞纠正镰刀状细胞疾病的基因突变。

1. 用诱导多能干细胞纠正镰刀状细胞疾病的基因突变
【动态】
倘若致病的基因突变能够被纠正，具有单基因突变的人体 诱导多能干细胞在模拟疾病表型，筛选候选药物和细胞替代疗法方面将有很大潜力。美国科学家最近报道了用基于同源重组的方式用两个突变的贝塔球蛋白等位基因在源自病人的诱导多能干细胞中精确纠正镰刀状细胞疾病的基因突变，在分化成红细胞时25-40%能够表达野生型产物。这个数据说明用人体诱导多能干细胞对人类基因组进行单核苷酸替换是可行的。

【点评】
该研究试图在人体诱导多能干细胞中对基因进行单核苷酸替换以纠正致病的基因突变。为因单基因突变导致的疾病提供了一种潜在的治疗策略。但是其前景依赖于诱导多能干细胞本身是否能用于临床治疗。

【参考论文】
Blood, 2011; DOI: 10.1182/blood-2011-02-335554
Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease
J. Zou, P. Mali, X. Huang, S. N. Dowey, L. Cheng.
Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here we report a homologous recombination (HR) based approach to precisely correct the Sickle Cell Disease (SCD) mutation in patient-derived iPSCs with two mutated beta-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate HR at the βs locus, we achieved precise conversion of one mutated βs to the wildtype βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected βs/βA heterozygous iPSCs that express at 25-40% level of the wildtype transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

2. 腺苷酸活化蛋白激酶乙酰化控制酵母内源性衰老
【动态】
组蛋白与非组蛋白的乙酰化是一种重要的翻译后修饰，影响了许多细胞进程。台湾和美国的科学家最近发现腺苷酸活化蛋白激酶（AMPK，AMP-activated protein kinase）中一个亚基Sip2能通过NuA4乙酰化，并随着细胞衰老，其乙酰化程度降低，从而提出Sip2乙酰化具有抗衰老的作用。 Sip2是Snf1复合物（AMPK）中的一种调控性β亚基，这项研究发现乙酰转移酶，和Rpd3去乙酰化酶能调控Sip2的乙酰化，并且Sip2乙酰化后，能与Snf1更加紧密的结合，增强Snf1复合物的催化活性，Sip2-Snf1的相互作用会抑制Snf1的活性，因而会降低下游靶标Sch9的磷酸化程度，并最终减缓生长的速度，延长生命周期。 进一步的实验证明Sip2的抗衰老作用与营养摄入和TORC1活性无关，从这些研究数据，研究人员提出了一种调控Sch9活性的蛋白乙酰化-磷酸化级联效应，这种效应能调控酵母内源性衰老，延长生命周期。

【点评】
该项表观调控的研究对于进一步了解衰老的机制有重要意义，也揭示了一种与营养摄入和TORC1活性无关的抗衰老途径。

【参考论文】
Cell, 2011, 146(6) pp. 969 - 979
Acetylation of Yeast AMPK Controls Intrinsic Aging Independently of Caloric Restriction

Jin-Ying Lu, Yu-Yi Lin, Jin-Chuan Sheu, et al.
Highlights
The yeast AMPK β subunit Sip2 is acetylated by NuA4 and deacetylated by Rpd3
Sip2 acetylation decreases with age, and increasing Sip2 acetylation extends life span
Acetylated Sip2 binds and inhibits Snf1, reducing Sch9 phosphorylation
The anti-aging effect of Sip2 acetylation is independent of nutrition and TORC activity
Summary
Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

3. 肠促胰岛素增强胰岛生命力
【动态】
在喂食情况下，肠促胰岛素GLP-1通过启动贝塔细胞中的cAMP途径来提升胰岛生命力。PKA活性的升高能刺激CREB的磷酸化，进而通过上调IRS2的表达增强贝塔细胞生存能力。尽管持久的GLP-1作用对于其对胰岛的有益的效果很重要，CREB活化的短暂性说明在此过程中有细胞核内因素的参与。随着快速诱导CREB调节的基因，cAMP启动了第二个通过HIF转录因子进行的基因表达期。cAMP的增加通过激活mTOR途径促进了HIF1α在贝塔细胞中的积累。正如暴露于雷帕霉素搞乱了GLP-1对贝塔细胞生命力的影响，该研究结果证明与肿瘤生长有关的一个生物学途径如何调节肠促胰岛素对胰岛功能的有益作用。

【点评】
肠分泌的降血糖激素GLP-1能够提高胰岛贝塔细胞的生命力，增加胰岛素的分泌。该研究进一步阐明了作用机制。同时说明恢复和提高衰老胃肠的分泌功能，对于预防和治疗糖尿病有重要意义。

【参考论文】
PNAS, September 26, 2011 DOI: 10.1073/pnas.1114228108
Inaugural Article: mTOR links incretin signaling to HIF induction in pancreatic beta cells
Sam Van de Velde, Meghan F. Hogan, and Marc Montminy.
Under feeding conditions, the incretin hormone GLP-1 promotes pancreatic islet viability by triggering the cAMP pathway in beta cells. Increases in PKA activity stimulate the phosphorylation of CREB, which in turn enhances beta cell survival by upregulating IRS2 expression. Although sustained GLP-1 action appears important for its salutary effects on islet function, the transient nature of CREB activation has pointed to the involvement of additional nuclear factors in this process. Following the acute induction of CREB-regulated genes, cAMP triggers a second delayed phase of gene expression that proceeds via the HIF transcription factor. Increases in cAMP promote the accumulation of HIF1α in beta cells by activating the mTOR pathway. As exposure to rapamycin disrupts GLP-1 effects on beta cell viability, these results demonstrate how a pathway associated with tumor growth also mediates salutary effects of an incretin hormone on pancreatic islet function.

4. 神经细胞生存的关键
【动态】
刺激突触内的NMDA受体诱导神经保护，而突触外的NMDA受体促进兴奋性毒性的细胞死亡。突触内的NMDA受体增强神经细胞表达环氧合酶-2（COX-2），尽管该酶调节神经功能，它也被看作是神经炎症的关键调节子，被认为通过过量生产前列腺素加剧兴奋性细胞毒性。这就造成明显悖论：突触内的NMDA受体是支持存活的但又是神经细胞大量表达COX-2所必须的。美国科学家就此假设刺激突触外的NMDA受体会将COX-2信号从一个生理学过程转变为一个可能的病理学过程。进一步的研究发现突触内外的NMDA受体以不同方式调节COX-2的表达和作用，突触内的NMDA受体增强COX-2的表达但持久的突触刺激通过降低细胞内COX-2的主要底物花生四烯酸的水平限制了COX-2的作用，相反，突触外的NMDA受体降低COX-2的表达但激活磷脂酶A2通过水解膜磷脂提高花生四烯酸水平。因此，顺序激活突触内然后突触外的NMDA受体会最大化COX-2依赖的前列腺素的合成。他们的研究也显示兴奋性细胞毒作用通过异常的突触网络兴奋性只诱导COX-2的表达，以及花生四烯酸和其他
多不饱和脂肪酸的非酶脂质过氧化是一种网络功能。该研究结果可能导致新的认识模式：与中风、癫痫和神经退化有关的病理学COX-2信号需要特定时空刺激NMDA受体。

【点评】
该研究表明在不同时空刺激同一种受体决定了神经细胞是存活还是死亡。

【参考论文】
Journal of Neuroscience, 2011; 31 (39): 13710 DOI: 10.1523/JNEUROSCI.3544-11.2011
Synaptic and Extrasynaptic NMDA Receptors Differentially Modulate Neuronal Cyclooxygenase-2 Function, Lipid Peroxidation, and Neuroprotection
D. T. Stark, N. G. Bazan.
Stimulation of synaptic NMDA receptors (NMDARs) induces neuroprotection, while extrasynaptic NMDARs promote excitotoxic cell death. Neuronal expression of cyclooxygenase-2 (COX-2) is enhanced by synaptic NMDARs, and although this enzyme mediates neuronal functions, COX-2 is also regarded as a key modulator of neuroinflammation and is thought to exacerbate excitotoxicity via overproduction of prostaglandins. This raises an apparent paradox: synaptic NMDARs are pro-survival yet are essential for robust neuronal COX-2 expression. We hypothesized that stimulation of extrasynaptic NMDARs converts COX-2 signaling from a physiological to a potentially pathological process. We combined HPLC-electrospray ionization-tandem MS-based mediator lipidomics and unbiased image analysis in mouse dissociated and organotypic cortical cultures to uncover that synaptic and extrasynaptic NMDARs differentially modulate neuronal COX-2 expression and activity. We show that synaptic NMDARs enhance neuronal COX-2 expression, while sustained synaptic stimulation limits COX-2 activity by suppressing cellular levels of the primary COX-2 substrate, arachidonic acid (AA). In contrast, extrasynaptic NMDARs suppress COX-2 expression while activating phospholipase A2, which enhances AA levels by hydrolysis of membrane phospholipids. Thus, sequential activation of synaptic then extrasynaptic NMDARs maximizes COX-2-dependent prostaglandin synthesis. We also show that excitotoxic events only drive induction of COX-2 expression through abnormal synaptic network excitability. Finally, we show that nonenzymatic lipid peroxidation of arachidonic and other polyunsaturated fatty acids is a function of network activity history. A new paradigm emerges from our results suggesting that pathological COX-2 signaling associated with models of stroke, epilepsy, and neurodegeneration requires specific spatiotemporal NMDAR stimulation.

5. 人体脂肪细胞脂质翻转造成代谢疾病
【动态】
脂肪细胞储存和清除甘油三酯决定了脂肪组织的重量。但是少为人知的是人体健康和疾病时的脂质翻转。一个国际合作团队对此进行了体内研究。通过测定脂肪细胞脂质中来自地上核试验的C14确定脂质的年龄，他们发现在人体脂肪细胞平均10年的寿命中，甘油三酯更新6次，脂质年龄与脂肪细胞大小无关，在很宽的年龄段的成人体内都是恒定的，且无性别差异。但是，脂肪细胞脂质翻转与脂质代谢紊乱有很强的关联。在肥胖时，甘油三酯清除率（脂肪分解和氧化）下降，每年储存的甘油三酯增加。相反，在不肥胖的患有常见的遗传性脂代谢紊乱家族性混合型高血脂症的病人中脂质的清除和储存率都下降。脂质清除率与脂肪细胞分解甘油三酯的能力正相关，与胰岛素抗性负相关。他们的数据支持如下机理：脂肪细胞对脂质的清除和储存在健康和疾病时的作用不同。甘油三酯的高存储和低清除促进脂肪组织积累和肥胖。而甘油三酯的存储和清除下降减少了其通过脂肪组织的中转，促进了脂代谢紊乱。

【点评】
相对于健康人，前期的2型糖尿病患者及家族性混合型高血脂症病人这些代谢疾病患者体内发生了脂质翻转，其脂肪细胞对甘油三酯的储存和清除能力下降。

【参考论文】
Nature, 25 September 2011; DOI:10.1038/nature10426
Dynamics of human adipose lipid turnover in health and metabolic disease
Peter Arner, Samuel Bernard, Mehran Salehpour, et al. 
Adipose tissue mass is determined by the storage and removal of triglycerides in adipocytes. Little is known, however, about adipose lipid turnover in humans in health and pathology. To study this in vivo, here we determined lipid age by measuring 14C derived from above ground nuclear bomb tests in adipocyte lipids. We report that during the average ten-year lifespan of human adipocytes, triglycerides are renewed six times. Lipid age is independent of adipocyte size, is very stable across a wide range of adult ages and does not differ between genders. Adipocyte lipid turnover, however, is strongly related to conditions with disturbed lipid metabolism. In obesity, triglyceride removal rate (lipolysis followed by oxidation) is decreased and the amount of triglycerides stored each year is increased. In contrast, both lipid removal and storage rates are decreased in non-obese patients diagnosed with the most common hereditary form of dyslipidaemia, familial combined hyperlipidaemia. Lipid removal rate is positively correlated with the capacity of adipocytes to break down triglycerides, as assessed through lipolysis, and is inversely related to insulin resistance. Our data support a mechanism in which adipocyte lipid storage and removal have different roles in health and pathology. High storage but low triglyceride removal promotes fat tissue accumulation and obesity. Reduction of both triglyceride storage and removal decreases lipid shunting through adipose tissue and thus promotes dyslipidaemia. We identify adipocyte lipid turnover as a novel target for prevention and treatment of metabolic disease.

6. 人脑神经细胞迁移通道及其在婴儿期的衰退
【动态】
很多成年动物大脑的室下区能产生大量新的神经细胞去往嗅球神经。沿着侧脑室壁，未成熟的子代神经细胞沿着切线方向的联线迁移合并成一个嘴侧迁移流将室下区连接到嗅球神经。相反的，成年人的室下区含有低增生的间隔层分开了室管膜外壁和星形胶质细胞的室周带。最初的报告发现在成年人嘴侧迁移流中有新的室下区增殖的细胞但几乎没有迁移中的未成熟神经细胞。相反，随后的研究表明在人的室下区和嘴侧迁移流中存在大量增殖和迁移。美国科学家最新的研究发现人类婴儿在18个月之前其室下区和嘴侧迁移流包含广阔的未成熟神经细胞迁移通道，但是与以前的报道相反的是，这种原始的活动在大一些的小孩中减弱了在成人后几乎绝迹。出乎意料的是，在这有限的神经形成的时间段内，并非所有室下区的新神经元都去往嗅球神经，还有一个去往前额皮质的主要迁移路线。总体看来，这些发现揭示了在人出生后早期的室下区和皮质有强大的垂直迁移的未成熟神经细胞流。这些迁移路线代表了影响婴儿的神经损伤的可能位置。

【点评】
该研究发现人脑中干细胞活性几乎只存在于出生后18个月内，并且有从室下区迁移到皮质的新的路线，这些代表了影响新生儿的大脑损伤的可能部位。

【参考论文】
Nature, 28 September 2011 DOI: 10.1038/nature10487
Corridors of migrating neurons in the human brain and their decline during infancy
Nader Sanai, Thuhien Nguyen, Rebecca A. Ihrie, et al. 
The subventricular zone of many adult non-human mammals generates large numbers of new neurons destined for the olfactory bulb. Along the walls of the lateral ventricles, immature neuronal progeny migrate in tangentially oriented chains that coalesce into a rostral migratory stream (RMS) connecting the subventricular zone to the olfactory bulb. The adult human subventricular zone, in contrast, contains a hypocellular gap layer separating the ependymal lining from a periventricular ribbon of astrocytes. Some of these subventricular zone astrocytes can function as neural stem cells in vitro, but their function in vivo remains controversial. An initial report found few subventricular zone proliferating cells and rare migrating immature neurons in the RMS of adult humans. In contrast, a subsequent study indicated robust proliferation and migration in the human subventricular zone and RMS. Here we find that the infant human subventricular zone and RMS contain an extensive corridor of migrating immature neurons before 18 months of age but, contrary to previous reports, this germinal activity subsides in older children and is nearly extinct by adulthood. Surprisingly, during this limited window of neurogenesis, not all new neurons in the human subventricular zone are destined for the olfactory bulb—we describe a major migratory pathway that targets the prefrontal cortex in humans. Together, these findings reveal robust streams of tangentially migrating immature neurons in human early postnatal subventricular zone and cortex. These pathways represent potential targets of neurological injuries affecting neonates.