A53T-ë±-synuclein overexpression mouse model signaling and striatal synaptic plasticity

Parkinson’s disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse. Parkinson’s disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse. Tissue was dissected from the brain of 6 months old (2 WT / 2 TgA / 2 TgB striata, 2 WT / 2 TgA / 2 TgB brainstems/midbrains, 2 WT / 2 TgA / 2 TgB cerebella) and of 18+ months old mice (4 WT / 2 TgA / 2 TgB striata, 6 WT / 4 TgA / 3 TgB brainstems/midbrains, 6 WT / 5 TgA / 4 TgB cerebella). Tissues from individual, particularly old mice up to 28 months age were included here to strengthen the definition of progression markers reflecting old age.帕金森病（Parkinson’s disease, PD）是老年人群中第二高发的神经退行性疾病，其致病诱因包括突触前蛋白α-突触核蛋白（alpha-synuclein, SNCA）表达上调，或是该蛋白发生A53T突变。该病的病理性特征为多巴胺能黑质纹状体投射神经元的选择性易损性。本研究使用两株过表达人源A53T-SNCA的小鼠模型，分别在6月龄和18月龄时开展实验，在未出现神经元变性的情况下研究纹状体功能异常，以解析疾病早期的发病机制。高效液相色谱分析纹状体神经递质含量的结果显示，多巴胺（dopamine, DA）水平与SNCA的表达量呈正相关，这一现象在SNCA缺陷小鼠中也有观测到。在DA水平升高的老龄A53T-SNCA过表达小鼠纹状体中，通过免疫印迹法和放射自显影技术检测到多巴胺受体DRD1A与DRD2出现反常的上调，该发现与囊泡释放异常的假说相符。通过微阵列技术开展的大规模转录组学研究，以及对Homer1、Cb1、Atf2和Pde7b转录水平变化的定量实时逆转录聚合酶链式反应（quantitative real-time RT-PCR）验证结果表明，突触后多巴胺应答反应呈进行性下降。作为上述变化的功能后果，老龄转基因小鼠的皮层纹状体脑片无法诱导长时程抑制，且旷场实验中观测到这些小鼠的自发运动能力出现隐匿性下降。综上，A53T-SNCA过表达小鼠中出现的神经传递功能异常与突触可塑性降低，反映了在出现明显神经元变性之前，突触核蛋白病所导致的基底节早期功能改变。因此，该小鼠模型可用于模拟帕金森病的临床前阶段。 帕金森病（Parkinson’s disease, PD）是老年人群中第二高发的神经退行性疾病，其致病诱因包括突触前蛋白α-突触核蛋白（alpha-synuclein, SNCA）表达上调，或是该蛋白发生A53T突变。该病的病理性特征为多巴胺能黑质纹状体投射神经元的选择性易损性。本研究使用两株过表达人源A53T-SNCA的小鼠模型，分别在6月龄和18月龄时开展实验，在未出现神经元变性的情况下研究纹状体功能异常，以解析疾病早期的发病机制。高效液相色谱分析纹状体神经递质含量的结果显示，多巴胺（dopamine, DA）水平与SNCA的表达量呈正相关，这一现象在SNCA缺陷小鼠中也有观测到。在DA水平升高的老龄A53T-SNCA过表达小鼠纹状体中，通过免疫印迹法和放射自显影技术检测到多巴胺受体DRD1A与DRD2出现反常的上调，该发现与囊泡释放异常的假说相符。通过微阵列技术开展的大规模转录组学研究，以及对Homer1、Cb1、Atf2和Pde7b转录水平变化的定量实时逆转录聚合酶链式反应（quantitative real-time RT-PCR）验证结果表明，突触后多巴胺应答反应呈进行性下降。作为上述变化的功能后果，老龄转基因小鼠的皮层纹状体脑片无法诱导长时程抑制，且旷场实验中观测到这些小鼠的自发运动能力出现隐匿性下降。综上，A53T-SNCA过表达小鼠中出现的神经传递功能异常与突触可塑性降低，反映了在出现明显神经元变性之前，突触核蛋白病所导致的基底节早期功能改变。因此，该小鼠模型可用于模拟帕金森病的临床前阶段。 我们从6月龄小鼠（野生型（wild type, WT）、TgA型、TgB型各2例的纹状体；野生型、TgA型、TgB型各2例的脑干/中脑；野生型、TgA型、TgB型各2例的小脑）以及18月龄以上小鼠（野生型4例、TgA型2例、TgB型2例的纹状体；野生型6例、TgA型4例、TgB型3例的脑干/中脑；野生型6例、TgA型5例、TgB型4例的小脑）的脑组织中分离获取组织样本。本研究纳入了年龄最高可达28月龄的单只老龄小鼠的组织样本，以强化对反映衰老进程的标志物的定义。