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. In adult rats that received unilateral intra-cortical injections of AAV expressing shRNA against PTEN, immunostaining for c-fos under resting conditions (home cage, HC) and after 1 h 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 phosphorylation of ERK1/2. SIGNIFICANCE STATEMENT: Deletion or knockdown of the tumor suppressor gene PTEN enables regenerative growth of adult CNS axons after injury, which is accompanied by enhanced recovery of function. Consequently, PTEN represents a potential target for therapeutic interventions to enhance recovery after CNS injury. Here we show that activity-dependent IEG induction is attenuated in PTEN-depleted neurons. These findings raise the intriguing possibility that functional recovery due to regenerative growth may be limited by the disruption of plasticity-related signaling pathways, and that recovery might be enhanced by restoring PTEN expression after regenerative growth has been achieved.

中文翻译：

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在成人神经元中，AAVshRNA介导的PTEN敲低减弱了依赖活性的立即早期基因诱导。

PTEN的基因缺失或敲低可使CNS轴突再生，增强损伤后完整轴突的萌发，并诱导未受伤的成年神经元从头生长。然而，未知的是，成熟神经元中的PTEN缺失如何改变神经元的生理学。作为解决这个问题的第一步，我们使用了免疫细胞化学作为活性依赖标记，以评估在齿状回的皮质神经元和颗粒细胞中PTEN敲低的后果。在接受单侧皮质内注射AAV的shRNA表达针对PTEN的成年大鼠中，在静息条件下（笼子，HC）对c-fos进行免疫染色，并且在探索新型富集环境（EE）1小时后，没有发现热点。暗示异常活动的c-fos表达。计数显示，在HC中，PTEN缺失区域的c-fos阳性神经元数量与HC对侧皮质的同源区域数量相近，并且对EE的c-fos诱导程度相似。但是，响应皮层高频刺激（HFS）的IEG诱导在PTEN缺失区域被减弱。在齿状回中具有AAVshRNA介导的PTEN缺失的大鼠中，在PTEN缺失的区域废除了用穿孔路径的HFS诱导的IEG c-fos和Arc。使用针对磷酸化S6（mTOR激活的下游标记）和磷酸化ERK1 / 2的磷酸化特异性抗体进行的免疫染色显示，PTEN缺失区域中S6磷酸化被废除，但保留了ERK1 / 2磷酸化的激活。重要声明：肿瘤抑制基因PTEN的缺失或敲除可使成年CNS轴突在损伤后再生生长，并伴有功能恢复。因此，PTEN代表了治疗干预措施的潜在目标，以增强中枢神经系统损伤后的恢复。在这里，我们显示在PTEN缺失的神经元中，依赖于活性的IEG诱导减弱。这些发现提出了一种有趣的可能性，即由于再生生长而引起的功能恢复可能会受到可塑性相关信号通路的破坏的限制，并且在达到再生生长后，通过恢复PTEN的表达可能会增强恢复。在这里，我们显示在PTEN缺失的神经元中，依赖于活性的IEG诱导减弱。这些发现提出了一种有趣的可能性，即由于再生生长而引起的功能恢复可能会受到可塑性相关信号通路的破坏的限制，并且在达到再生生长后，通过恢复PTEN的表达可能会增强恢复。在这里，我们显示在PTEN缺失的神经元中，依赖于活性的IEG诱导减弱。这些发现提出了一种有趣的可能性，即由于再生生长而引起的功能恢复可能会受到可塑性相关信号通路的破坏的限制，并且在达到再生生长后，通过恢复PTEN的表达可能会增强恢复。