In vivo AAV-mediated expression of calbindin-D₂₈k in rat basal forebrain cholinergic neurons.
In vivo AAV-mediated expression of calbindin-D₂₈k in rat basal forebrain cholinergic neurons.
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DOI：
10.1016/j.jneumeth.2012.09.021
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发表时间：
2013-01-15
影响因子：
3
通讯作者：
Geula, Changiz
中科院分区：
文献类型：
--
作者：
Nagykery, Nicholas;Terwilliger, Ernest F.;Geula, Changiz
关键词：
人和非人灵长类动物基底前脑胆碱能神经元（BFCNs）富含钙结合蛋白-D28 k（CB）。我们已经表明，在正常衰老过程中，BFCN的CB选择性丢失，这似乎使这些神经元易于在阿尔茨海默病中缠结形成和变性。我们先前的初步调查表明，啮齿动物BFCN没有CB。在这里，我们证实，大鼠胆碱乙酰转移酶丰富的BFCN是没有CB免疫反应。然后，我们描述了一种方法，腺相关病毒载体（AAV）诱导表达CB在大鼠BFCNs在体内。我们构建了携带在CMV启动子或神经元特异性烯醇化酶（NSE）启动子控制下的CB基因的AAV载体，以偏向神经元中的表达。两种载体导致CB在小鼠神经元培养物中的表达，以及在注射后的大鼠脑中的表达。AAV-NSE-CB导致神经元中更稳健的表达。将10 μl AAV-NSE-CB注射到苍白球内段和内囊内的BFCNs组分中，CB在84%的BFCNs中表达。在14天时表达最佳。注射AAV-NSE-LacZ导致稳健的β-半乳糖苷酶表达，但没有CB免疫反应性。我们的研究结果表明，使用NSE启动子导致神经元中基因的高表达，并且BFCN可以靶向表达在啮齿动物和灵长类动物脑中差异表达的基因。这些发现对人类BFCN的基因替代治疗也有重要意义。
The cholinergic neurons of the basal forebrain (BFCNs) in human and non-human primates are rich in the calcium binding protein calbindin-D28k (CB). We have shown a selective loss of CB from BFCNs in the course of normal aging, which appears to predispose these neurons to tangle formation and degeneration in Alzheimer’s disease. Our previous preliminary investigation demonstrated that rodent BFCNs are devoid of CB. Here we confirm that rat choline acetyltransferase-rich BFCNs are devoid of CB immunoreactivity. We then describe a method for adeno-associated viral vector (AAV) induced expression of CB in rat BFCNs in vivo. We constructed AAV vectors bearing the CB gene under the control of the CMV promoter, or neuron-specific enolase (NSE) promoter, to bias expression in neurons. Both vectors resulted in CB expression in mouse neuronal cultures, and in rat brain following injections. AAV-NSE-CB resulted in more robust expression in neurons. Injections of 10 μl of AAV-NSE-CB in the BFCNs component located within the internal segment of globus pallidus and internal capsule resulted in expression of CB in 84% of BFCNs. Expression was optimum at 14 days. Injections of AAV-NSE-LacZ resulted in robust β-galactosidase expression, but no CB immunoreactivity. Our results show that use of NSE promoter leads to high expression of genes in neurons and that the BFCNs can be targeted for expression of genes that are differentially expressed in the rodent and primate brains. These findings also have important implications for gene replacement therapy in human BFCNs.
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DOI：
10.3791/562
发表时间：
2007-01-01
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Hilgenberg, Lutz G W;Smith, Martin A
通讯作者：
Smith, Martin A
影响因子：
11.2
作者：
ROGERS, JD;BROGAN, D;MIRRA, SS
通讯作者：
MIRRA, SS
影响因子：
2.1
作者：
SAMUEL, WA;HENDERSON, VW;MILLER, CA
通讯作者：
MILLER, CA
DOI：
10.1093/jnen/62.6.605
发表时间：
2003-06-01
影响因子：
3.2
作者：
Geula, C;Nagykery, N;Bu, J
通讯作者：
Bu, J
影响因子：
2.9
作者：
D'Orlando, C;Fellay, B;Celio, MR
通讯作者：
Celio, MR