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。具体目的： 目标1：在野生型（WT）小鼠中证明依赖性的行为。目的1目标是确定依赖于静态活动的行为，并将使用具有遗传靶向的静脉体病变来实现。 AIM 2：确定体内脑膜体活动编码行为。目标2的目标是识别有效行为的曲折活动模式，并将使用体内钙成像来完成。 AIM 3：测试曲局的活性是否区分LRRK2突变小鼠。目的3目标是测试焦虑变化的焦虑和选择行为是否伴随着PD小鼠模型的改变，并将使用LRRK2 G2019S KI小鼠来实现。 2。科学前提和意义 尽管被认为是一种运动障碍，但PD还是重要的非运动症状的焦虑和判断力受损的症状。 SPN被分为静脉组和基质室。链球菌可能是支持PD中的情感和执行功能的候选者，因为脑膜炎SPN与边缘区域的连接比位于Matrix的边缘区域具有更强的连接，因此在压力和成本敏感的选择行为中具有新的作用，并且在应激和成本敏感的选择行为中起着作用，并且可以从核控制核心焦虑症中获得独特的支架。 PD中最常见的遗传因子是编码富含亮氨酸重复激酶2（LRRK2）的基因中的突变，该基因在脑室SPN中高度表达。 LRRK2 G2019S KI小鼠在认知和焦虑量的测量中表现出微妙的异常，可以模拟PD的非运动症状。超动态表型可能表明执行控制丧失。实际上，最近具有健康（风险敏感）或强迫性（不敏感）行为的小鼠的遗传筛查发现，重要的LRRK2上调会区分强迫动物。据报道，LRRK2 G2019S突变可增强LRRK2激酶活性，但未经测试，这种常见的PD连接突变对体内静脉体活性的影响。这项研究确定了焦虑和选择行为的神经动态，对心理健康具有重要意义。它对PD非常重要，因为从出生开始就存在LRRK2突变，并且非运动症状可能在PD发作之前。因此，与小鼠LRRK2突变相对应的神经生理学的任何差异都可以提供有关PD发展，早期诊断以及如何恢复生理以预防或治疗疾病的信息。这项研究有可能为LRRK2突变携带者定义新的病理生理表型，该表型可能是由新的预防性疗法和疾病治疗靶向的。 3。创新 这是对焦虑和选择表现中的神经元角色的创新研究，首先是在野生型小鼠中，后来在携带LRRK2 G2019S错过突变的小鼠中。概念创新存在于对静脉曲炎神经元和PD的联合关注中，以及直接关注为非运动PD的神经激活的关注。这是由于技术创新的微型头部安装显微镜和遗传编码的荧光钙传感器的可能性，因此可以揭示清醒小鼠的动态神经活动。 4。研究计划 在AIM 1中，使用SEPW1_NP67（SEPW1）小鼠在静脉体体中表达CRE，我们双侧注射了纹状体内AAV表达CRE依赖性的diphtheria toxin a Fragment（AAV-DTA），以产生遗传型的骨膜组。我们将比较病变和假对照，以测试候选行为的依赖性。开放式测试评估基本运动，零迷宫（EZM）和轻/暗盒评估焦虑。固定间隔30秒（FI30）操作食品自我管理评估了在标准和奖励评估条件下的积极选择绩效。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AIM 2中，我们分别使用SEPW1或矩阵-CRE小鼠（PLXND1-OG1）小鼠引入了具有CRE依赖性荧光钙传感器GCAMP6S或基质的AAV，并分别使用植入植入的Miniscops进行的自定义Miniscops在植入的Miniscop上进行植入的Miniscop，并在植入的小鼠中进行光学记录的神经活动。我们将静脉体组神经元之间的活动与AIM 1（例如杠杆或区域转换）研究的相同候选行为的事件相关联，并将其与基质神经元之间的活动进行比较。成像是由三只SEPW1小鼠引起的，表明链球菌弱编码开阔场的速度，并且较大的静脉曲组激活伴随着（大概是更高的）向光中的过渡，而不是光/黑暗盒子测试中的黑暗区域。当小鼠导航光/黑暗盒子时，神经活动可以与区域之间过渡的精确时间保持一致。通过这种方式，我们可以鉴定在这些过渡事件中可靠活跃的单个曲折神经元。细胞图表明可以为每只小鼠制作已识别神经元的位置，并且可能有助于识别参与神经元的解剖趋势。我们发现，许多过渡带来的神经元没有替代过渡。这表明不同的静脉曲炎神经元可能对区域转变具有独特的选择性。最后，我们发现，与区域内的净行为相比，探索区域（前过渡）探索区域（过渡前）的行为之间的相关性更大。在行为数据中，我们看到小鼠花费更多的时间探索光，而不是每个过渡的黑暗区域，尤其是在早期试验中。这种对过渡的犹豫措施可能表现出焦虑。同时，当犹豫最高时，我们的初步数据表明，相对于黑暗探索的光探索相应的探索过程中的激活更大。 在AIM 3中，为了测试Striosome活性是否区分LRRK2突变小鼠，我们将重复具有G2019S KI突变及其LRRK2-WT的sepw1-cre小鼠中静脉体组动力学的体内成像。我们将比较突变体和WT动物之间的行为和体内静脉体活性，以确定与PD相关的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的显着上调，表明神经兴奋性改变了。 G2019S突变被称为增强LRRK2活性64,66。 AIM 3测试这是否会影响曲局的神经元激活。