AAVshRNA-mediated PTEN knockdown in adult neurons attenuates activity-dependent immediate early gene induction.
AAVshRNA-mediated PTEN knockdown in adult neurons attenuates activity-dependent immediate early gene induction.
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DOI：
10.1016/j.expneurol.2019.113098
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发表时间：
2020-04
影响因子：
5.3
通讯作者：
Yee KM
中科院分区：
文献类型：
--
作者：
Steward O;Coulibaly AP;Metcalfe M;Yonan JM;Yee KM
基因缺失或敲低PTEN能够使CNS轴突再生，增强损伤后完整轴突的发芽，并诱导未损伤的成年神经元从头生长。然而，成熟神经元中的PTEN缺失如何改变神经元生理学尚不清楚。作为解决这个问题的第一步，我们使用免疫细胞化学的活性依赖性标记物，以评估的后果，在皮质神经元和颗粒细胞的齿状回的PTEN敲低。成年大鼠接受单侧皮质内注射表达针对PTEN的shRNA的AAV，并允许存活2个月。在静息条件下（饲养笼，HC）和探索新的富集环境（EE）1小时后，c-fos的免疫染色显示没有提示异常活性的c-fos表达热点。计数显示，在HC中，PTEN缺失区与对侧皮质中的同源区中的c-fos阳性神经元的数量相似，并且与EE相似的c-fos诱导。然而，在对皮质的高频刺激（HFS）的反应中，IEG诱导在PTEN缺失区中减弱。在具有AAVshRNA介导的齿状回中的PTEN缺失的大鼠中，在PTEN缺失的区域中，具有穿通路径的HFS的IEGs c-fos和Arc的诱导被废除。使用磷酸化-S6（mTOR活化的下游标记物）和磷酸化-ERK 1/2的磷酸化特异性抗体的免疫染色揭示了在PTEN缺失区域中S6磷酸化的废除，但保留了ERK 1/2的磷酸化活化。
Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. Adult rats received unilateral intra-cortical injections of AAV expressing shRNA against PTEN and were allowed to survive for 2 months. Immunostaining for c-fos under resting conditions (home cage, HC) and after 1hr of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphosphorylation of ERK1/2.
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影响因子：
5.3
作者：
Gallent EA;Steward O
通讯作者：
Steward O
影响因子：
4.8
作者：
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Bito H
影响因子：
5.3
作者：
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通讯作者：
Steward, Oswald
影响因子：
5.3
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通讯作者：
Blackmore, Murray G.
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5.3
作者：
Williams, Michael R.;DeSpenza, Tyrone, Jr.;Luikart, Bryan W.
通讯作者：
Luikart, Bryan W.