Characterization of Non-motor Phenotypes in Parkinson's Disease

帕金森病非运动表型的特征

• 批准号: 10250914
• 负责人: Huaibin Cai
• 金额: $ 5.14万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 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/10250914
• 关键词: Address; Affect; Affective; Aging; Alpha-Synuclein transgenic mouse; Anatomy; Animals; Anxiety; Area; Attention; Behavior; Bilateral; Birth; Bradykinesia; Brain; Calcium; Caliber; Cell Nucleus; Cells; Choice Behavior; Clinical; Cognition; Cognition Disorders; Cognitive; Corpus striatum structure; Custom; Data; Dependence; Development; Disease; Early Diagnosis; Emotional disorder; Event; Food; Functional disorder; Gene Expression; Genes; Genetic; Genetic Screening; Head; Hippocampus (Brain); Histology; Image; Impaired cognition; Impairment; Implant; Individual; Judgment; Knock-in Mouse; Knockout Mice; LRRK2 gene; Lesion; Light; Link; Locomotion; Maps; Measures; Mental Health; Microscope; Midbrain structure; Missense Mutation; Modeling; Motor; Motor Activity; Movement Disorders; Mus; Mutant Strains Mice; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Onset of illness; Optics; Parkinson Disease; Pattern; Performance; Phenotype; Phosphotransferases; Physiology; Positioning Attribute; Potassium Channel; Proteins; Reporting; Research; Rest Tremor; Rewards; Risk; Role; Self Administration; Stress; Symptoms; System; Testing; Time; Up-Regulation; Wild Type Mouse; alpha synuclein; awake; behavior test; behavioral study; cognitive function; cohort; cost; diphtheria toxin fragment A; disability; dopaminergic neuron; emotional functioning; executive function; experimental study; field study; frontal lobe; in vivo; in vivo calcium imaging; in vivo imaging; innovation; interest; lens; mouse model; mutant; mutation carrier; nerve supply; neurophysiology; non-motor symptom; posture instability; prevent; prophylactic; relating to nervous system; selective expression; sensor; striosome; trend

1. Specific Aims: Aim 1: Demonstrate striosome-dependent behavior in wildtype (WT) mice. The Aim 1 objective is to identify behaviors reliant on striosome activity, and will be accomplished using genetically targeted striosome lesions. Aim 2: Identify in vivo striosomal activity encoding behavior. The Aim 2 objective is to identify striosome activity patterns serving behavior, and will be accomplished using in vivo calcium imaging. Aim 3: Test whether striosome activity distinguishes Lrrk2 mutant mice. The Aim 3 objective is to test whether altered striosome dynamics accompany altered anxiety and choice behaviors in PD mouse models, and will be accomplished using Lrrk2 G2019S KI mice. 2. Scientific Premise and Significance Despite recognition as a movement disorder, PD includes important non-motor symptoms of elevated anxiety and impaired judgement. SPNs are divided into striosome and matrix compartments. Striosomes are a likely candidate to support affective and executive functions impaired in PD, since striosomal SPNs have stronger connections to limbic regions than those located in matrix, have newly indicated roles in stress- and cost-sensitive choice behaviors, and receive unique innervation from a nucleus controlling anxiety. The single most common genetic factor in PD is mutation in the gene encoding Leucine-rich repeat kinase 2 (LRRK2), which is highly expressed in striosomal SPNs. Lrrk2 G2019S KI mice show subtle abnormality in measures of cognition and anxiety that could model non-motor symptoms of PD. A hyper-kinetic phenotype may indicate a loss of executive control. Indeed, a recent genetic screen of mice with healthy (risk-sensitive) or else compulsive (risk-insensitive) behavior found that significant Lrrk2 upregulation distinguishes compulsive animals. LRRK2 G2019S mutation is reported to enhance LRRK2 kinase activity, but the influence of this common PD-linked mutation on striosome activity in vivo is untested. This research identifies neural dynamics underlying anxiety and choice behaviors with major significance for mental health. It is highly significant to PD as LRRK2 mutation is present from birth, and non-motor symptoms may precede PD onset. Therefore, any difference in neurophysiology corresponding to Lrrk2 mutation in mice could provide information on PD development, early diagnosis, and how physiology might be restored to prevent or treat disease. This research has potential to define a new pathophysiological phenotype for LRRK2 mutation carriers which may be targeted by new prophylactic therapies and disease treatments. 3. Innovation This is an innovative study of striosome neuron roles in anxiety and choice performance, first in wild-type mice and later in mice carrying Lrrk2 G2019S missense mutation. Conceptual innovation exists in the combined focus on striosome neurons and PD, and in the direct focus on neural activation serving non-motor PD. This is possible due to technically innovative miniature head-mounted microscopes and genetically encoded fluorescent calcium sensors to reveal dynamic neural activity in awake mice. 4. Research Plan In Aim 1, using Sepw1_NP67 (Sepw1) mice expressing Cre in striosomes, we bilaterally injected intra-striatal AAV expressing Cre-dependent diphtheria toxin A fragment (AAV-DTA) to create genetically targeted striosome lesions. We will compare lesioned and sham controls to test candidate behaviors for striosome-dependence. Open-field test assesses basic locomotion, and Elevated Zero Maze (EZM) and Light/Dark Box assess anxiety. Fixed-Interval 30second (FI30) operant food self-administration assesses motivated choice performance under standard and reward-devalued conditions. One cohort of animals has completed Aim 1 experiments, demonstrating the feasibility of the behavioral tests. Data from this cohort will be analyzed once histology confirms the lesion. In Aim 2, we introduced AAV with Cre-dependent fluorescent calcium sensor GCaMP6s to striosomes or matrix using Sepw1 or matrix-Cre mice (Plxnd1-OG1) mice, respectively, and optically recorded neural activity in behaving mice using custom MiniScopes above an implanted 1mm diameter GRIN lens. We related activity among striosome neurons to events in the same candidate behaviors studied in Aim 1, such as lever-press or area-transition, and will compare this to activity among matrix neurons. Imaging results from three Sepw1 mice suggest that striosomes weakly encode velocity in the Open-field, and that greater striosome activation accompanies the (presumably higher-anxiety) transitions in to the Light as opposed to Dark areas in the Light/Dark Box test. As mice navigate the Light/Dark box, neural activity can be aligned to the precise time of transition between areas. In this way we can identify individual striosome neurons which are reliably active (positive z-score) at these transition events. Cell maps indicating the position of identified neurons can be made for each mouse, and may be useful for identifying anatomical trends in engaged neurons. We find that many neurons engaged by one transition are not engaged by alternative transitions. This suggests that distinct striosome neurons may have distinct selectivity for area transitions. Finally, we find greater correlation between striosome neural activation and the behavior of exploring an area (pre-transition) as opposed to net behavior within an area. In behavior data, we see mice spend more time exploring the Light as opposed to the Dark area per transition, particularly in early trials. This measure of hesitation to transition may demonstrate anxiety. At the same time when hesitation is highest, our preliminary data suggest greater striosome activation during corresponding exploration of Light relative to exploration of Dark. In Aim 3, to test whether striosome activity distinguishes Lrrk2 mutant mice, we will repeat Aim 2 in vivo imaging of striosome dynamics in Sepw1-Cre mice with G2019S KI mutation and in their Lrrk2-WT littermates. We will compare behavior and in vivo striosome activity between mutant and WT animals to determine effects of PD-related Lrrk2 G2019S KI mutation. Our gene expression studies found significant upregulation in potassium channels and channel associated proteins in Lrrk2 KO mice, suggesting altered neural excitability. G2019S mutation is purported to enhance Lrrk2 activity 64,66. Aim 3 tests whether this influences striosome neuron activation.

1. 具体目标： 目标 1：展示野生型 (WT) 小鼠的纹状体依赖性行为。目标 1 的目标是识别依赖纹状体活性的行为，并将通过基因靶向纹状体损伤来实现。 目标 2：识别体内纹状体活性编码行为。目标 2 的目标是识别服务于行为的纹状体活动模式，并将使用体内钙成像来实现。 目标 3：测试纹状体活性是否能够区分 Lrrk2 突变小鼠。 Aim 3 的目标是测试 PD 小鼠模型中纹状体动力学的改变是否伴随着焦虑和选择行为的改变，并将使用 Lrrk2 G2019S KI 小鼠来完成。 2 科学前提及意义 尽管帕金森病被认为是一种运动障碍，但它包括焦虑加剧和判断力受损等重要的非运动症状。 SPN 分为纹状体和基质区室。纹状体很可能是支持帕金森病中情感和执行功能受损的候选者，因为纹状体 SPN 与边缘区域的联系比位于基质中的那些更强，在压力和成本敏感的选择行为中具有新的作用，并从控制焦虑的核心。 PD 中最常见的单一遗传因素是编码富含亮氨酸重复激酶 2 (LRRK2) 的基因突变，该基因在纹状体 SPN 中高度表达。 Lrrk2 G2019S KI 小鼠在认知和焦虑方面表现出微妙的异常，可以模拟 PD 的非运动症状。过度运动表型可能表明执行控制的丧失。事实上，最近对具有健康（风险敏感）或强迫（风险不敏感）行为的小鼠进行的基因筛查发现，显着的 Lrrk2 上调可以区分强迫动物。据报道，LRRK2 G2019S 突变可增强 LRRK2 激酶活性，但这种常见的 PD 相关突变对体内纹状体活性的影响尚未经过测试。这项研究确定了焦虑和选择行为背后的神经动力学，对心理健康具有重大意义。这对 PD 非常重要，因为 LRRK2 突变从出生起就存在，并且非运动症状可能先于 PD 发病。因此，小鼠中与 Lrrk2 突变相对应的神经生理学差异可以提供有关 PD 发展、早期诊断以及如何恢复生理学以预防或治疗疾病的信息。这项研究有可能为 LRRK2 突变携带者定义一种新的病理生理表型，这可能是新的预防疗法和疾病治疗的目标。 3、创新 这是一项关于纹状体神经元在焦虑和选择表现中的作用的创新研究，首先在野生型小鼠中进行，随后在携带 Lrrk2 G2019S 错义突变的小鼠中进行。概念创新存在于对纹状体神经元和帕金森病的联合关注，以及对服务于非运动帕金森病的神经激活的直接关注。这是可能的，因为技术创新的微型头戴式显微镜和基因编码的荧光钙传感器可以揭示清醒小鼠的动态神经活动。 四、研究计划 在目标 1 中，使用在纹状体中表达 Cre 的 Sepw1_NP67 (Sepw1) 小鼠，我们双侧注射纹状体内表达 Cre 依赖性白喉毒素 A 片段 (AAV-DTA) 的 AAV，以产生遗传靶向纹状体损伤。我们将比较受损对照和假对照，以测试候选行为的纹状体依赖性。旷场测试评估基本运动，高架零迷宫 (EZM) 和明/暗盒测试评估焦虑。固定间隔 30 秒 (FI30) 操作性食物自我管理评估标准和奖励贬值条件下的动机选择表现。一组动物已经完成了 Aim 1 实验，证明了行为测试的可行性。一旦组织学证实病变，将分析该队列的数据。 在目标 2 中，我们分别使用 Sepw1 或基质-Cre 小鼠 (Plxnd1-OG1) 小鼠将具有 Cre 依赖性荧光钙传感器 GCaMP6 的 AAV 引入到纹状体或基质中，并使用植入的 1mm 上方的定制 MiniScopes 光学记录行为小鼠的神经活动直径梯度折射率透镜。我们将纹状体神经元之间的活动与目标 1 中研究的相同候选行为中的事件相关联，例如杠杆按压或区域转换，并将其与基质神经元之间的活动进行比较。三只 Sepw1 小鼠的成像结果表明，纹状体在开放场中对速度的编码较弱，并且在明/暗盒测试中，纹状体的激活伴随着向光区（可能是较高焦虑）的转变，而不是向暗区的转变。当小鼠在明/暗盒中导航时，神经活动可以与区域之间转换的精确时间对齐。通过这种方式，我们可以识别在这些转换事件中可靠活跃（正 z 分数）的单个纹状体神经元。可以为每只小鼠制作指示已识别神经元位置的细胞图，并且可能有助于识别参与神经元的解剖趋势。我们发现许多参与一种转换的神经元并未参与其他转换。这表明不同的纹状体神经元可能对区域转换具有不同的选择性。最后，我们发现纹状体神经激活与探索区域（过渡前）的行为之间的相关性比区域内的净行为更大。在行为数据中，我们看到小鼠在每次转换时花费更多时间探索光区而不是暗区，特别是在早期试验中。这种对转型的犹豫可能会表现出焦虑。与此同时，当犹豫程度最高时，我们的初步数据表明，相对于探索黑暗，在相应的光探索中纹状体激活程度更高。 在目标 3 中，为了测试纹状体活性是否能区分 Lrrk2 突变小鼠，我们将在具有 G2019S KI 突变的 Sepw1-Cre 小鼠及其 Lrrk2-WT 同窝小鼠中重复目标 2 的纹状体动力学体内成像。我们将比较突变体和 WT 动物之间的行为和体内纹状体活性，以确定 PD 相关 Lrrk2 G2019S KI 突变的影响。我们的基因表达研究发现 Lrrk2 KO 小鼠中钾通道和通道相关蛋白显着上调，表明神经兴奋性发生改变。 G2019S 突变据称可增强 Lrrk2 活性 64,66。目标 3 测试这是否影响纹状体神经元激活。