世界生命科學(xué)前沿動態(tài)周報（五十九）

（9.26-10.2/2011）
美寶國際集團:陶國新 

主要內(nèi)容：用誘導(dǎo)多能干細胞糾正鐮刀狀細胞疾病的基因突變；活化蛋白激酶乙?；刂平湍竷?nèi)源性衰老；腸促胰島素增強胰島生命力；神經(jīng)細胞生存的關(guān)鍵；人體脂肪細胞脂質(zhì)翻轉(zhuǎn)造成代謝疾病；人腦神經(jīng)細胞遷移通道及其在嬰兒期的衰退。

焦點動態(tài)：用誘導(dǎo)多能干細胞糾正鐮刀狀細胞疾病的基因突變。

1. 用誘導(dǎo)多能干細胞糾正鐮刀狀細胞疾病的基因突變
【動態(tài)】
倘若致病的基因突變能夠被糾正，具有單基因突變的人體 誘導(dǎo)多能干細胞在模擬疾病表型，篩選候選藥物和細胞替代療法方面將有很大潛力。美國科學(xué)家最近報道了用基于同源重組的方式用兩個突變的貝塔球蛋白等位基因在源自病人的誘導(dǎo)多能干細胞中精確糾正鐮刀狀細胞疾病的基因突變，在分化成紅細胞時25-40%能夠表達野生型產(chǎn)物。這個數(shù)據(jù)說明用人體誘導(dǎo)多能干細胞對人類基因組進行單核苷酸替換是可行的。

【點評】
該研究試圖在人體誘導(dǎo)多能干細胞中對基因進行單核苷酸替換以糾正致病的基因突變。為因單基因突變導(dǎo)致的疾病提供了一種潛在的治療策略。但是其前景依賴于誘導(dǎo)多能干細胞本身是否能用于臨床治療。

【參考論文】
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內(nèi)源性衰老
【動態(tài)】
組蛋白與非組蛋白的乙?；且环N重要的翻譯后修飾，影響了許多細胞進程。臺灣和美國的科學(xué)家最近發(fā)現(xiàn)腺苷酸活化蛋白激酶（AMPK，AMP-activated protein kinase）中一個亞基Sip2能通過NuA4乙?；?，并隨著細胞衰老，其乙酰化程度降低，從而提出Sip2乙?；哂锌顾ダ系淖饔?。 Sip2是Snf1復(fù)合物（AMPK）中的一種調(diào)控性β亞基，這項研究發(fā)現(xiàn)乙酰轉(zhuǎn)移酶，和Rpd3去乙酰化酶能調(diào)控Sip2的乙?；⑶襍ip2乙?；?，能與Snf1更加緊密的結(jié)合，增強Snf1復(fù)合物的催化活性，Sip2-Snf1的相互作用會抑制Snf1的活性，因而會降低下游靶標Sch9的磷酸化程度，并最終減緩生長的速度，延長生命周期。 進一步的實驗證明Sip2的抗衰老作用與營養(yǎng)攝入和TORC1活性無關(guān)，從這些研究數(shù)據(jù)，研究人員提出了一種調(diào)控Sch9活性的蛋白乙酰化-磷酸化級聯(lián)效應(yīng)，這種效應(yīng)能調(diào)控酵母內(nèi)源性衰老，延長生命周期。

【點評】
該項表觀調(diào)控的研究對于進一步了解衰老的機制有重要意義，也揭示了一種與營養(yǎng)攝入和TORC1活性無關(guān)的抗衰老途徑。

【參考論文】
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. 腸促胰島素增強胰島生命力
【動態(tài)】
在喂食情況下，腸促胰島素GLP-1通過啟動貝塔細胞中的cAMP途徑來提升胰島生命力。PKA活性的升高能刺激CREB的磷酸化，進而通過上調(diào)IRS2的表達增強貝塔細胞生存能力。盡管持久的GLP-1作用對于其對胰島的有益的效果很重要，CREB活化的短暫性說明在此過程中有細胞核內(nèi)因素的參與。隨著快速誘導(dǎo)CREB調(diào)節(jié)的基因，cAMP啟動了第二個通過HIF轉(zhuǎn)錄因子進行的基因表達期。cAMP的增加通過激活mTOR途徑促進了HIF1α在貝塔細胞中的積累。正如暴露于雷帕霉素搞亂了GLP-1對貝塔細胞生命力的影響，該研究結(jié)果證明與腫瘤生長有關(guān)的一個生物學(xué)途徑如何調(diào)節(jié)腸促胰島素對胰島功能的有益作用。

【點評】
腸分泌的降血糖激素GLP-1能夠提高胰島貝塔細胞的生命力，增加胰島素的分泌。該研究進一步闡明了作用機制。同時說明恢復(fù)和提高衰老胃腸的分泌功能，對于預(yù)防和治療糖尿病有重要意義。

【參考論文】
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. 神經(jīng)細胞生存的關(guān)鍵
【動態(tài)】
刺激突觸內(nèi)的NMDA受體誘導(dǎo)神經(jīng)保護，而突觸外的NMDA受體促進興奮性毒性的細胞死亡。突觸內(nèi)的NMDA受體增強神經(jīng)細胞表達環(huán)氧合酶-2（COX-2），盡管該酶調(diào)節(jié)神經(jīng)功能，它也被看作是神經(jīng)炎癥的關(guān)鍵調(diào)節(jié)子，被認為通過過量生產(chǎn)前列腺素加劇興奮性細胞毒性。這就造成明顯悖論：突觸內(nèi)的NMDA受體是支持存活的但又是神經(jīng)細胞大量表達COX-2所必須的。美國科學(xué)家就此假設(shè)刺激突觸外的NMDA受體會將COX-2信號從一個生理學(xué)過程轉(zhuǎn)變?yōu)橐粋€可能的病理學(xué)過程。進一步的研究發(fā)現(xiàn)突觸內(nèi)外的NMDA受體以不同方式調(diào)節(jié)COX-2的表達和作用，突觸內(nèi)的NMDA受體增強COX-2的表達但持久的突觸刺激通過降低細胞內(nèi)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，突觸外的NMDA受體降低COX-2的表達但激活磷脂酶A2通過水解膜磷脂提高花生四烯酸水平。因此，順序激活突觸內(nèi)然后突觸外的NMDA受體會最大化COX-2依賴的前列腺素的合成。他們的研究也顯示興奮性細胞毒作用通過異常的突觸網(wǎng)絡(luò)興奮性只誘導(dǎo)COX-2的表達，以及花生四烯酸和其他
多不飽和脂肪酸的非酶脂質(zhì)過氧化是一種網(wǎng)絡(luò)功能。該研究結(jié)果可能導(dǎo)致新的認識模式：與中風(fēng)、癲癇和神經(jīng)退化有關(guān)的病理學(xué)COX-2信號需要特定時空刺激NMDA受體。

【點評】
該研究表明在不同時空刺激同一種受體決定了神經(jīng)細胞是存活還是死亡。

【參考論文】
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. 人體脂肪細胞脂質(zhì)翻轉(zhuǎn)造成代謝疾病
【動態(tài)】
脂肪細胞儲存和清除甘油三酯決定了脂肪組織的重量。但是少為人知的是人體健康和疾病時的脂質(zhì)翻轉(zhuǎn)。一個國際合作團隊對此進行了體內(nèi)研究。通過測定脂肪細胞脂質(zhì)中來自地上核試驗的C14確定脂質(zhì)的年齡，他們發(fā)現(xiàn)在人體脂肪細胞平均10年的壽命中，甘油三酯更新6次，脂質(zhì)年齡與脂肪細胞大小無關(guān)，在很寬的年齡段的成人體內(nèi)都是恒定的，且無性別差異。但是，脂肪細胞脂質(zhì)翻轉(zhuǎn)與脂質(zhì)代謝紊亂有很強的關(guān)聯(lián)。在肥胖時，甘油三酯清除率（脂肪分解和氧化）下降，每年儲存的甘油三酯增加。相反，在不肥胖的患有常見的遺傳性脂代謝紊亂家族性混合型高血脂癥的病人中脂質(zhì)的清除和儲存率都下降。脂質(zhì)清除率與脂肪細胞分解甘油三酯的能力正相關(guān)，與胰島素抗性負相關(guān)。他們的數(shù)據(jù)支持如下機理：脂肪細胞對脂質(zhì)的清除和儲存在健康和疾病時的作用不同。甘油三酯的高存儲和低清除促進脂肪組織積累和肥胖。而甘油三酯的存儲和清除下降減少了其通過脂肪組織的中轉(zhuǎn)，促進了脂代謝紊亂。

【點評】
相對于健康人，前期的2型糖尿病患者及家族性混合型高血脂癥病人這些代謝疾病患者體內(nèi)發(fā)生了脂質(zhì)翻轉(zhuǎn)，其脂肪細胞對甘油三酯的儲存和清除能力下降。

【參考論文】
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. 人腦神經(jīng)細胞遷移通道及其在嬰兒期的衰退
【動態(tài)】
很多成年動物大腦的室下區(qū)能產(chǎn)生大量新的神經(jīng)細胞去往嗅球神經(jīng)。沿著側(cè)腦室壁，未成熟的子代神經(jīng)細胞沿著切線方向的聯(lián)線遷移合并成一個嘴側(cè)遷移流將室下區(qū)連接到嗅球神經(jīng)。相反的，成年人的室下區(qū)含有低增生的間隔層分開了室管膜外壁和星形膠質(zhì)細胞的室周帶。最初的報告發(fā)現(xiàn)在成年人嘴側(cè)遷移流中有新的室下區(qū)增殖的細胞但幾乎沒有遷移中的未成熟神經(jīng)細胞。相反，隨后的研究表明在人的室下區(qū)和嘴側(cè)遷移流中存在大量增殖和遷移。美國科學(xué)家最新的研究發(fā)現(xiàn)人類嬰兒在18個月之前其室下區(qū)和嘴側(cè)遷移流包含廣闊的未成熟神經(jīng)細胞遷移通道，但是與以前的報道相反的是，這種原始的活動在大一些的小孩中減弱了在成人后幾乎絕跡。出乎意料的是，在這有限的神經(jīng)形成的時間段內(nèi)，并非所有室下區(qū)的新神經(jīng)元都去往嗅球神經(jīng)，還有一個去往前額皮質(zhì)的主要遷移路線。總體看來，這些發(fā)現(xiàn)揭示了在人出生后早期的室下區(qū)和皮質(zhì)有強大的垂直遷移的未成熟神經(jīng)細胞流。這些遷移路線代表了影響嬰兒的神經(jīng)損傷的可能位置。

【點評】
該研究發(fā)現(xiàn)人腦中干細胞活性幾乎只存在于出生后18個月內(nèi)，并且有從室下區(qū)遷移到皮質(zhì)的新的路線，這些代表了影響新生兒的大腦損傷的可能部位。

【參考論文】
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.