Effects of a null mutation of the tumor suppressor gene PTEN in dopamine neurons

抑癌基因 PTEN 无效突变对多巴胺神经元的影响

• 批准号: 7966876
• 负责人: Cristina Backman
• 金额: $ 38.95万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7966876
• 关键词: Ablation; Acute; Address; Adult; Age; Aging; Alleles; Animals; Apoptosis; Apoptotic; Area; Behavioral; Brain; Breeding; Cells; Cerebral cortex; Chromosomes, Human, Pair 10; Cocaine; Cognition; Complex; Corpus striatum structure; Development; Dopamine; Dopamine D1 Receptor; Dopamine D2 Receptor; Dorsal; Dose; Down-Regulation; Drug abuse; Embryo; Environment; Exposure to; Fiber; Gene Expression; Genes; Genetic; Genomics; Hippocampus (Brain); Homologous Gene; Human; Hypertrophy; Lead; Lesion; Link; Longevity; Malignant Neoplasms; Marijuana; Mediating; Messenger RNA; Microdialysis; Midbrain structure; Mitosis; Modeling; Modification; Molecular; Motivation; Mus; Mutant Strains Mice; Mutation; NR1 gene; Neurodegenerative Disorders; Neurons; Neurotransmitter Receptor; Neurotransmitters; Nicotine; Organism; PTEN gene; Parkinson Disease; Pathway interactions; Peptides; Performance; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Play; Property; Proteins; Rattus; Reporting; Research Personnel; Resistance; Rewards; Role; Schedule; Serotonin; Signal Pathway; Social Interaction; Staging; Stem cells; Substantia nigra structure; Surface; System; Therapeutic; Time; Tissues; Transgenic Model; Tumor Suppressor Genes; Tumor Suppressor Proteins; Ventral Tegmental Area; addiction; aged; cognitive function; congenic; dopamine system; dopamine transporter; dopaminergic neuron; drug of abuse; extracellular; insight; mutant; neuroadaptation; neurotoxic; neurotransmission; nigrostriatal system; null mutation; preference; preproenkephalin; prevent; prodynorphin; receptor; response; sensory stimulus; serotonin 5 receptor; tensin; treatment strategy; tumor; young adult

The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinsons disease, and other neurodegenerative disorders. PTEN deletion or Akt/PKB activation in dopamine neurons of the ventral midbrain results in remarkable hypertrophy of the substantia nigra and VTA. Our initial characterization of a DAT-PTEN KO strain has provided a clear definition of some of the neuroadaptations in the mesolimbic and nigrostriatal systems, and clearly show dopamine neurotransmission is permanently altered in PTEN KO mice. However, while DAT-PTEN KO animals are viable and appear behaviorally competent, an in depth study of behavioral parameters will clarify if the lack of PTEN interferes with essential functions related to the dopamine system in young, adult and aged animals. Studies performed over the past few years have clearly shown that phenotypes caused by specific genetic modifications are strongly influenced by genes unlinked to the target locus. This problem is exacerbated through the use of Cre-LoxP models as two strains, often containing their own (obscure) genetic backgrounds, are crossed through very specific breeding schedules to generate control and experimental animals. Clearly, it becomes important to avoid the use of a mixed genetic background so complex as to preclude any reasonable use of controls and prevent replication by other investigators. To perform complex behavioral studies, we will use a c57bl/6 congenic DAT-PTEN KO mouse line, generated in our lab. A new mechanism in the brain of rats that may mediate the rewarding and reinforcing properties of drugs of abuse has recently been discovered. This mechanism involves the physical interaction between two proteins in midbrain dopamine neurons, the tumor suppressor PTEN and the brain specific receptor for serotonin, the 5-HT2c receptor (5-HT2cR). Blocking the interaction of PTEN with 5-HT2cR prevents the development of conditioned place preference to nicotine and marijuana. In addition, PTEN has been shown to physically interact with the NR1 subunit of NMDAR in hippocampus, and PTEN downregulation decreases NMDAR surface expression. These studies suggest PTEN in dopamine neurons may directly modulate functions intimately linked to the development of addiction, and dopamine mediated cognition, such as responses to reward and motivation. We will use a congenic PTEN-KO line to analyze in detail the behavioral profile of KO animals in relationship to drug abuse, overall locomotor performance, as well as other dopamine related cognitive functions. We have shown Pten deletion in differentiated DA neurons causes a significant increase in the number and size of surviving neurons in both the mesolimbic and nigrostriatal projecting pathways (see above). Because at the time of Pten deletion DA neurons have already completed mitosis and phenotypic determination, it is unlikely the reported increase in DA neurons is due to an increase in newly formed neurons. It is thus likely PTEN ablation preserves DA neurons that normally would undergo apoptosis due to the lack of target support, by repressing the initiation of apoptotic pathways. We are now intrigued about several aspects induced by PTEN deletion in dopamine cells: Do all dopamine neurons preserved in DAT-PTEN KO animals project to target areas and form functional connections? Can exposure to an enriched environment enhance dopaminergic function in young and aged KO animals? Do the mesolimbic and nigrostriatal dopamine projections remain functional into aging? Is the PTENless aging dopaminergic system more or less resistant to neurotoxic insults applied during different stages of the mouse life span? Are PTENless dopamine neurons prone to form tumors? Obviously, the tremendous adaptations observed in the PTENless dopaminergic system during development, may pose problems for interpretation of results; however, as previous studies have shown Akt/PKB activation in the adult dopaminergic system can also result in remarkable hypertrophy and plasticity of the nigra and VTA, the results obtained in this study may provide valuable insights into how PTEN ablation changes dopamine function at the molecular and behavioral levels, and the long-term consequences of such adaptations. We believe these studies are important, as manipulations of the PTEN pathway are being considered as a possible venue for therapeutic strategies involving the brain.

由此产生的突变小鼠表现出神经元肥大，腹侧中脑多巴胺能神经元和纤维数量增加。 有趣的是，Pten KO小鼠的定量微透析研究显示，尽管总DA组织水平显著增加，但基础DA细胞外水平或背侧纹状体中诱发的DA释放没有改变。KO动物的纹状体多巴胺受体D1（DRD 1）和强啡肽原（PDyn）mRNA水平显著升高，表明与纹状体黑质投射通路相关的神经元活动增强，而多巴胺受体D2（DRD 2）和前脑啡肽原（PPE）mRNA水平保持不变。此外，PTEN失活保护DA神经元，并显着增强DA依赖性行为功能的KO小鼠进行性6 OHDA损伤后。这些结果提供了关于PTEN在大脑中的作用的进一步证据，并表明在发育过程中对PTEN/Akt信号通路的操纵可能改变多巴胺能神经传递的基础状态，并可能为治疗帕金森病和其他神经退行性疾病提供治疗策略。 腹侧中脑多巴胺神经元中的PTEN缺失或Akt/PKB激活导致黑质和VTA显著肥大。我们对DAT-PTEN KO菌株的初步表征提供了中脑边缘和黑质纹状体系统中一些神经适应的明确定义，并清楚地表明在PTEN KO小鼠中多巴胺神经传递被永久改变。然而，虽然DAT-PTEN KO动物是可行的，并且表现出行为能力，但对行为参数的深入研究将澄清是否缺乏PTEN会干扰与年轻，成年和老年动物中多巴胺系统相关的基本功能。过去几年进行的研究清楚地表明，由特定遗传修饰引起的表型受到与目标基因座无关的基因的强烈影响。通过使用Cre-LoxP模型，这一问题变得更加严重，因为两个菌株通常包含自己的（模糊的）遗传背景，通过非常特定的育种计划进行杂交，以产生对照和实验动物。显然，避免使用混合遗传背景变得很重要，因为这种背景太复杂，以至于无法合理使用对照，并阻止其他研究者进行复制。为了进行复杂的行为研究，我们将使用在我们实验室中产生的c57 bl/6同源DAT-PTEN KO小鼠系。 最近在老鼠大脑中发现了一种新的机制，它可以调节滥用药物的奖励和强化特性。 该机制涉及中脑多巴胺神经元中的两种蛋白质之间的物理相互作用，肿瘤抑制因子PTEN和5-羟色胺的脑特异性受体5-HT 2c受体（5-HT 2cR）。阻断PTEN与5-HT 2cR的相互作用可以防止对尼古丁和大麻的条件性位置偏好的发展。此外，已显示PTEN与海马中NMDAR的NR 1亚基物理相互作用，并且PTEN下调降低NMDAR表面表达。这些研究表明，多巴胺神经元中的PTEN可能直接调节与成瘾发展密切相关的功能，以及多巴胺介导的认知，例如对奖励和动机的反应。 我们将使用同源的PTEN-KO系来详细分析KO动物的行为特征与药物滥用、整体运动表现以及其他多巴胺相关的认知功能的关系。 我们已经表明，在分化的DA神经元中Pten缺失导致中脑边缘和黑质纹状体投射通路中存活神经元的数量和大小显著增加（见上文）。因为在Pten缺失时，DA神经元已经完成有丝分裂和表型确定，所以报告的DA神经元增加不太可能是由于新形成的神经元增加。因此，可能的是，PTEN消融通过抑制凋亡途径的起始来保留通常由于缺乏靶支持而经历凋亡的DA神经元。我们现在对多巴胺细胞中PTEN缺失诱导的几个方面很感兴趣：DAT-PTEN KO动物中保存的所有多巴胺神经元是否都投射到靶区域并形成功能连接？暴露于丰富的环境中能增强年轻和老年KO动物的多巴胺能功能吗？中脑边缘和黑质纹状体的多巴胺投射是否在衰老过程中仍然起作用？在小鼠寿命的不同阶段，无PTEN的衰老多巴胺能系统对神经毒性损伤的抵抗力是多还是少？没有PTEN的多巴胺神经元容易形成肿瘤吗？显然，在发育过程中，在无PTEN的多巴胺能系统中观察到的巨大适应可能会给结果的解释带来问题;然而，如先前的研究所示，成人多巴胺能系统中的Akt/PKB活化也可导致黑质和腹侧被盖区的显著肥大和可塑性，本研究中获得的结果可以提供关于PTEN切除如何在分子和行为水平上改变多巴胺功能的有价值的见解，以及这种适应的长期后果。我们相信这些研究是重要的，因为PTEN通路的操纵被认为是涉及大脑的治疗策略的可能场所。