Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Lon...Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2 4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light （470 nm）, and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.展开更多
We have delivered viral vectors containing either Chop2 fused with GFP, Channelrhodopsin-2 （ChR2）, or Halorhodopsin （HaloR） fused with mCherry （to form light gated cation channels or chloride pumps, respectively...We have delivered viral vectors containing either Chop2 fused with GFP, Channelrhodopsin-2 （ChR2）, or Halorhodopsin （HaloR） fused with mCherry （to form light gated cation channels or chloride pumps, respectively）, into the dorsal cochlear nucleus （DCN）. One to eighteen months later we examined the CN and inferior colliculus （IC） for evidence of virally transfected cells and processes. Production of ChR2 and HaloR was observed throughout the DCN. Rhodopsin localization within neurons was determined, with elongate, fusiform and giant cells identified based on morphology and location within the DCN. Production of ChR2 and HaloR was found at both the injection site as well as in regions projecting to and from the DCN. Light driven neuronal activity in the DCN was dependent upon the wavelength and intensity of the light, with only the appropriate wavelength resulting in activation and higher intensity light resulting in more neuronal activity. Transfecting cells via viral delivery of rhodopsins can be useful as a tract tracer and as a neuronal marker to delineate pathways. In the future rhodopsin delivery and activation may be developed as an alternative to electrical stimulation of neurons.展开更多
背景程序性细胞死亡配体1(programmed cell death ligand-1,PD-L1)和配体2(programmed cell death ligand-2,PD-L2)与程序性细胞死亡受体1(programmed cell death protein-1,PD-1)的相互作用是一个介导免疫逃逸的免疫抑制检查点,因此是...背景程序性细胞死亡配体1(programmed cell death ligand-1,PD-L1)和配体2(programmed cell death ligand-2,PD-L2)与程序性细胞死亡受体1(programmed cell death protein-1,PD-1)的相互作用是一个介导免疫逃逸的免疫抑制检查点,因此是癌症中基于阻滞的免疫治疗的重要靶点。在非小细胞肺癌(nonsmall-celllungcancer,NSCLC)中,有必要对PD-1检查点阻滞反应生物学进行深入了解,并确定生物标志物以预测其对免疫疗法的临床反应。在本研究中,我们系统描述了NSCLC中PD-L1和PD-L2表达相关基因。方法我们进行了回顾性比较分析,来确定NSCLC中PD-L1和PD-L2 mRNA表达相关基因。为此,我们考察了肿瘤细胞系百科全书(Cancer Cell Line Encyclopedia,CCLE)数据库中肺–非小细胞(lung non-small-cell,Lung_NSC)和癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中肺腺癌(lung adenocarcinoma,LUAD)和鳞状细胞癌(lung squamous cell carcinoma,LUSC)的可用数据集。结果通过对CCLE数据集Lung_NSC的分析确定了PD-L1和PD-L2之间的表达相关性。此外,我们发现了489个基因与PD-L1相关、191个基因与PD-L2相关,以及111个基因与二者均有表达相关性。在TCGA数据集LUAD和LUSC中对PD-L1和PD-L2也进行了表达相关研究。在LUAD中,我们发现了257个基因与PD-L1相关、914个基因与PD-L2相关以及211个基因与二者均有表达相关性。在LUSC中,我们发现了26个基因与PD-L1相关、326个基因与PD-L2相关以及13个基因与二者均有表达相关性。只有少数基因表达在CCLE和TCGA数据集中均表现为与PD-L1和PD-L2相关。涉及干扰素信号转导基因的表达尤其与Lung_NSC中的PD-L1、LUSC中的PD-L2以及LUAD中的PD-L1和PD-L2的表达相关基因汇聚。在LUSC,PD-L1的表达,以及PD-L2的表达(相比之下相关程度较小)与染色体9p24区的基因相关,表明染色体9p24拓扑相关结构域是LUSC中PD-L1表达特别重要的驱动力。对PD-L1和PD-L2的受体PD-1、分化群80(cluster of differentiation,CD80)和排斥导向分子B(repulsive guidance molecule B,RGMB)的表达相关分析表明,在LUAD中PD-1和CD80表达与PD-L1和PD-L2均相关。在LUSC中CD80表达与PD-L2相关。结论我们提出了与NSCLC中PD-L1和PD-L2 mRNA表达相关的基因特征,这可能对于了解PD-1检查点阻滞反应生物学和开发基于基因特征的生物标志物以预测免疫疗法的临床反应具有重要意义。展开更多
To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 m...To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.展开更多
目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系...目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系有光感基因组(n=21)。HE染色观察膀胱组织的病理学改变,Real time PCR和Western blotting检测膀胱逼尿肌受体P2X1、P2X3在分子和蛋白水平的表达情况。结果在建模过程中,诱发电位监测发现术中有3只动物脊髓受损,其余动物建模成功。与脊髓栓系无光感基因组比较,在HE染色结果中脊髓栓系有光感基因组逼尿肌细胞形态大小相对均一,肌纤维排列较有序,趋近于对照组。Real time PCR和Western blotting检测结果显示,与对照组比较,脊髓栓系无光感基因组P2X1、P2X3 mRNA和相对蛋白表达量均升高,差异有统计学意义(P<0.05);而经蓝光照射一定时间后,脊髓栓系有光感基因组中两种受体mRNA和相对蛋白表达量均降低,与脊髓栓系无光感基因组比较,差异有统计学意义(P<0.05);与对照组比较,差异无统计学意义(P>0.05)。结论光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3有一定的调控作用。展开更多
Background:Programmed cell death ligand-1(PD-L1)and ligand-2(PD-L2)interaction with programmed cell death protein-1(PD-1)represent an immune-inhibiting checkpoint mediating immune evasion and is,accordingly,an importa...Background:Programmed cell death ligand-1(PD-L1)and ligand-2(PD-L2)interaction with programmed cell death protein-1(PD-1)represent an immune-inhibiting checkpoint mediating immune evasion and is,accordingly,an important target for blockade-based immunotherapy in cancer.In non-small-cell lung cancer(NSCLC),improved understanding of PD-1 checkpoint blockade-responsive biology and identification of biomarkers for prediction of a clinical response to immunotherapy is warranted.Thus,in the present study,we systematically described PD-L1 and PD-L2 expression correlated genes in NSCLC.Methods:We performed comparative retrospective analyses to identify PD-L1 and PD-L2 mRNA expression corre-lated genes in NSCLC.For this,we examined available datasets from the cancer cell line encyclopedia(CCLE)project lung non-small-cell(Lung_NSC)and the cancer genome atlas(TCGA)projects lung adenocarcinoma(LUAD)and squamous cell carcinoma(LUSC).Results:Analysis of the CCLE dataset Lung_NSC identified expression correlation between PD-L1 and PD-L2.Moreo-ver,we identified expression correlation between 489 genes and PD-L1,191 genes and PD-L2,and 111 genes for both.PD-L1 and PD-L2 also expression correlated in TCGA datasets LUAD and LUSC.In LUAD,we identified expression corre-lation between 257 genes and PD-L1,914 genes and PD-L2,and 211 genes for both.In LUSC,we identified expression correlation between 26 genes and PD-L1,326 genes and PD-L2,and 13 genes for both.Only a few genes expression correlated with PD-L1 and PD-L2 across the CCLE and TCGA datasets.Expression of Interferon signaling-involved genes converged in particular with the expression correlated genes for PD-L1 in Lung_NSC,for PD-L2 in LUSC,and for both PD-L1 and PD-L2 in LUAD.In LUSC,PD-L1,and to a lesser extent PD-L2,expression correlated with chromosome 9p24 localized genes,indicating a chromosome 9p24 topologically associated domain as an important driver of in particu-lar LUSC PD-L1 expression.Expression correlation analyses of the PD-L1 and PD-L2 receptors programmed cell death protein-1(PD-1),Cluster of differentiation 80(CD80),and Repulsive guidance molecule B(RGMB)showed that PD-1 and CD80 expression correlated with both PD-L1 and PD-L2 in LUAD.CD80 expression correlated with PD-L2 in LUSC.Conclusions:We present gene signatures associated with PD-L1 and PD-L2 mRNA expression in NSCLC which could possess importance in relation to understand PD-1 checkpoint blockade-responsive biology and development of gene signature based biomarkers for predicting clinical responses to immunotherapy.展开更多
Optogenetics is a combination of optics and genetics technology that can be used to activate or inhibit specific cells in tissues. It has been used to treat Parkinson’s disease, epilepsy and neurological diseases, bu...Optogenetics is a combination of optics and genetics technology that can be used to activate or inhibit specific cells in tissues. It has been used to treat Parkinson’s disease, epilepsy and neurological diseases, but rarely Alzheimer’s disease. Adeno-associated virus carrying the CaMK promoter driving the optogenetic channelrhodopsin-2 (CHR2) gene (or without the CHR2 gene, as control) was injected into the bilateral dentate gyri, followed by repeated intrahippocampal injections of soluble low-molecular-weight amyloid-β1–42 peptide (Aβ1–42). Subsequently, the region was stimulated with a 473 nm laser (1–3 ms, 10 Hz, 5 minutes). The novel object recognition test was conducted to test memory function in mice. Immunohistochemical staining was performed to analyze the numbers of NeuN and synapsin Ia/b-positive cells in the hippocampus. Western blot assay was carried out to analyze the expression levels of glial fibrillary acidic protein, NeuN, synapsin Ia/b, metabotropic glutamate receptor-1a (mGluR-1a), mGluR-5, N-methyl-D-aspartate receptor subunit NR1, glutamate receptor 2, interleukin-1β, interleukin-6 and interleukin-10. Optogenetic stimulation improved working and short-term memory in mice with Alzheimer’s disease. This neuroprotective effect was associated with increased expression of NR1, glutamate receptor 2 and mGluR-5 in the hippocampus, and decreased expression of glial fibrillary acidic protein and interleukin-6. Our results show that optogenetics can be used to regulate the neuronal-glial network to ameliorate memory functions in mice with Alzheimer’s disease. The study was approved by the Animal Resources Committee of Jinan University, China (approval No. LL-KT-2011134) on February 28, 2011.展开更多
The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remod...The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.展开更多
基金sponsored by the National Natural Science Foundation of China (General Program),No.81070749,31070882,30970758Key Project of Chongqing Science & Technology Commission,No. 2010AB5118
文摘Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2 4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light （470 nm）, and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.
基金supported by Ralph Wilson Foundation(to A.G.H)Capita Foundation(to A.G.H)
文摘We have delivered viral vectors containing either Chop2 fused with GFP, Channelrhodopsin-2 （ChR2）, or Halorhodopsin （HaloR） fused with mCherry （to form light gated cation channels or chloride pumps, respectively）, into the dorsal cochlear nucleus （DCN）. One to eighteen months later we examined the CN and inferior colliculus （IC） for evidence of virally transfected cells and processes. Production of ChR2 and HaloR was observed throughout the DCN. Rhodopsin localization within neurons was determined, with elongate, fusiform and giant cells identified based on morphology and location within the DCN. Production of ChR2 and HaloR was found at both the injection site as well as in regions projecting to and from the DCN. Light driven neuronal activity in the DCN was dependent upon the wavelength and intensity of the light, with only the appropriate wavelength resulting in activation and higher intensity light resulting in more neuronal activity. Transfecting cells via viral delivery of rhodopsins can be useful as a tract tracer and as a neuronal marker to delineate pathways. In the future rhodopsin delivery and activation may be developed as an alternative to electrical stimulation of neurons.
文摘背景程序性细胞死亡配体1(programmed cell death ligand-1,PD-L1)和配体2(programmed cell death ligand-2,PD-L2)与程序性细胞死亡受体1(programmed cell death protein-1,PD-1)的相互作用是一个介导免疫逃逸的免疫抑制检查点,因此是癌症中基于阻滞的免疫治疗的重要靶点。在非小细胞肺癌(nonsmall-celllungcancer,NSCLC)中,有必要对PD-1检查点阻滞反应生物学进行深入了解,并确定生物标志物以预测其对免疫疗法的临床反应。在本研究中,我们系统描述了NSCLC中PD-L1和PD-L2表达相关基因。方法我们进行了回顾性比较分析,来确定NSCLC中PD-L1和PD-L2 mRNA表达相关基因。为此,我们考察了肿瘤细胞系百科全书(Cancer Cell Line Encyclopedia,CCLE)数据库中肺–非小细胞(lung non-small-cell,Lung_NSC)和癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中肺腺癌(lung adenocarcinoma,LUAD)和鳞状细胞癌(lung squamous cell carcinoma,LUSC)的可用数据集。结果通过对CCLE数据集Lung_NSC的分析确定了PD-L1和PD-L2之间的表达相关性。此外,我们发现了489个基因与PD-L1相关、191个基因与PD-L2相关,以及111个基因与二者均有表达相关性。在TCGA数据集LUAD和LUSC中对PD-L1和PD-L2也进行了表达相关研究。在LUAD中,我们发现了257个基因与PD-L1相关、914个基因与PD-L2相关以及211个基因与二者均有表达相关性。在LUSC中,我们发现了26个基因与PD-L1相关、326个基因与PD-L2相关以及13个基因与二者均有表达相关性。只有少数基因表达在CCLE和TCGA数据集中均表现为与PD-L1和PD-L2相关。涉及干扰素信号转导基因的表达尤其与Lung_NSC中的PD-L1、LUSC中的PD-L2以及LUAD中的PD-L1和PD-L2的表达相关基因汇聚。在LUSC,PD-L1的表达,以及PD-L2的表达(相比之下相关程度较小)与染色体9p24区的基因相关,表明染色体9p24拓扑相关结构域是LUSC中PD-L1表达特别重要的驱动力。对PD-L1和PD-L2的受体PD-1、分化群80(cluster of differentiation,CD80)和排斥导向分子B(repulsive guidance molecule B,RGMB)的表达相关分析表明,在LUAD中PD-1和CD80表达与PD-L1和PD-L2均相关。在LUSC中CD80表达与PD-L2相关。结论我们提出了与NSCLC中PD-L1和PD-L2 mRNA表达相关的基因特征,这可能对于了解PD-1检查点阻滞反应生物学和开发基于基因特征的生物标志物以预测免疫疗法的临床反应具有重要意义。
基金supported by National Science Foundation of China(31571091 to Mingliang Pu),National Basic Research Program of China (2015CB351806 to Mingliang Pu)National Institutes of Health Grant (NIH) (EY17130 to Zhuo-Hua Pan)Dryer Foundation, the Ligon Research Center of Vision, and Research to Prevent Blindness to Department of Ophthalmology at Wayne State University
文摘To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.
文摘目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系有光感基因组(n=21)。HE染色观察膀胱组织的病理学改变,Real time PCR和Western blotting检测膀胱逼尿肌受体P2X1、P2X3在分子和蛋白水平的表达情况。结果在建模过程中,诱发电位监测发现术中有3只动物脊髓受损,其余动物建模成功。与脊髓栓系无光感基因组比较,在HE染色结果中脊髓栓系有光感基因组逼尿肌细胞形态大小相对均一,肌纤维排列较有序,趋近于对照组。Real time PCR和Western blotting检测结果显示,与对照组比较,脊髓栓系无光感基因组P2X1、P2X3 mRNA和相对蛋白表达量均升高,差异有统计学意义(P<0.05);而经蓝光照射一定时间后,脊髓栓系有光感基因组中两种受体mRNA和相对蛋白表达量均降低,与脊髓栓系无光感基因组比较,差异有统计学意义(P<0.05);与对照组比较,差异无统计学意义(P>0.05)。结论光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3有一定的调控作用。
基金This study was supported by Familien Erichsens Familiefond.TVL and DH are supported by Ph.D.fellowships from the Faculty of Health,Aarhus University,Denmark
文摘Background:Programmed cell death ligand-1(PD-L1)and ligand-2(PD-L2)interaction with programmed cell death protein-1(PD-1)represent an immune-inhibiting checkpoint mediating immune evasion and is,accordingly,an important target for blockade-based immunotherapy in cancer.In non-small-cell lung cancer(NSCLC),improved understanding of PD-1 checkpoint blockade-responsive biology and identification of biomarkers for prediction of a clinical response to immunotherapy is warranted.Thus,in the present study,we systematically described PD-L1 and PD-L2 expression correlated genes in NSCLC.Methods:We performed comparative retrospective analyses to identify PD-L1 and PD-L2 mRNA expression corre-lated genes in NSCLC.For this,we examined available datasets from the cancer cell line encyclopedia(CCLE)project lung non-small-cell(Lung_NSC)and the cancer genome atlas(TCGA)projects lung adenocarcinoma(LUAD)and squamous cell carcinoma(LUSC).Results:Analysis of the CCLE dataset Lung_NSC identified expression correlation between PD-L1 and PD-L2.Moreo-ver,we identified expression correlation between 489 genes and PD-L1,191 genes and PD-L2,and 111 genes for both.PD-L1 and PD-L2 also expression correlated in TCGA datasets LUAD and LUSC.In LUAD,we identified expression corre-lation between 257 genes and PD-L1,914 genes and PD-L2,and 211 genes for both.In LUSC,we identified expression correlation between 26 genes and PD-L1,326 genes and PD-L2,and 13 genes for both.Only a few genes expression correlated with PD-L1 and PD-L2 across the CCLE and TCGA datasets.Expression of Interferon signaling-involved genes converged in particular with the expression correlated genes for PD-L1 in Lung_NSC,for PD-L2 in LUSC,and for both PD-L1 and PD-L2 in LUAD.In LUSC,PD-L1,and to a lesser extent PD-L2,expression correlated with chromosome 9p24 localized genes,indicating a chromosome 9p24 topologically associated domain as an important driver of in particu-lar LUSC PD-L1 expression.Expression correlation analyses of the PD-L1 and PD-L2 receptors programmed cell death protein-1(PD-1),Cluster of differentiation 80(CD80),and Repulsive guidance molecule B(RGMB)showed that PD-1 and CD80 expression correlated with both PD-L1 and PD-L2 in LUAD.CD80 expression correlated with PD-L2 in LUSC.Conclusions:We present gene signatures associated with PD-L1 and PD-L2 mRNA expression in NSCLC which could possess importance in relation to understand PD-1 checkpoint blockade-responsive biology and development of gene signature based biomarkers for predicting clinical responses to immunotherapy.
基金supported by the National Natural Science Foundation of China,No.81171191(to LYZ)the Shenzhen Special Fund Project on Strategic Emerging Industry Development of China,No.JCYJ20160422170522075(to LYZ)the Shenzhen Healthcare Research Project of China,No.201601015(to LYZ)
文摘Optogenetics is a combination of optics and genetics technology that can be used to activate or inhibit specific cells in tissues. It has been used to treat Parkinson’s disease, epilepsy and neurological diseases, but rarely Alzheimer’s disease. Adeno-associated virus carrying the CaMK promoter driving the optogenetic channelrhodopsin-2 (CHR2) gene (or without the CHR2 gene, as control) was injected into the bilateral dentate gyri, followed by repeated intrahippocampal injections of soluble low-molecular-weight amyloid-β1–42 peptide (Aβ1–42). Subsequently, the region was stimulated with a 473 nm laser (1–3 ms, 10 Hz, 5 minutes). The novel object recognition test was conducted to test memory function in mice. Immunohistochemical staining was performed to analyze the numbers of NeuN and synapsin Ia/b-positive cells in the hippocampus. Western blot assay was carried out to analyze the expression levels of glial fibrillary acidic protein, NeuN, synapsin Ia/b, metabotropic glutamate receptor-1a (mGluR-1a), mGluR-5, N-methyl-D-aspartate receptor subunit NR1, glutamate receptor 2, interleukin-1β, interleukin-6 and interleukin-10. Optogenetic stimulation improved working and short-term memory in mice with Alzheimer’s disease. This neuroprotective effect was associated with increased expression of NR1, glutamate receptor 2 and mGluR-5 in the hippocampus, and decreased expression of glial fibrillary acidic protein and interleukin-6. Our results show that optogenetics can be used to regulate the neuronal-glial network to ameliorate memory functions in mice with Alzheimer’s disease. The study was approved by the Animal Resources Committee of Jinan University, China (approval No. LL-KT-2011134) on February 28, 2011.
基金supported by the National Key Research and Development Program of China (2016YFA0500801 to Xinjian He)
文摘The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.
