自发罹患帕金森病食蟹猴模型

我们发现一例疑似自然发生 PD 的 10 岁（接近人类 30-40 岁）雄性食蟹猴，其 PD 临床症状典型，包括运动迟缓、静止性震颤与姿势障碍；左旋多巴与阿扑吗啡的治疗效果类似 PD 患者的表现；病理染色表明黑质多巴胺能神经元形态损伤明显，数量丢失显著（~70%）；胞内磷酸化 alpha-synuclein 聚集增多，以及路易突起，黑质小胶质细胞与星形胶质细胞的增生与激活显著。而且，此猴脑脊液多巴胺含量也显著低于对照，其发病机理可能与 LRRK2 突变有关。参考超过 1500 只食蟹猴的 11 个 PD 风险基因测序结果，此猴 LRRK2 与 ATP13A2 基因出现了罕见的错义突变，体外功能验证表明，LRRK2 突变可能促进了PD 病理发生（如磷酸化 LRRK2 升高、磷酸化 Rab10 升高，RNA-seq表明激活的通路涉及免疫异常与炎症反应）。该工作首次证实：除了人类，猴子也会自然发生 PD，其发病年龄模拟了人类早发 PD 的特征，而 LRRK2 突变也体现了 PD 患者“gain-of-function”的潜在发病机理。
数据质量：a. 实验动物和伦理
实验方案和动物福利均获得中国科学院昆明动物研究所伦理委员会的批准（IACUC No. 20041）。猕猴（包括恒河猴与食蟹猴）单笼饲养，标准的明/暗周期，自由取食饮水，由兽医照看。共 60 只成年猴参与自发 PD 的筛选。
b. 行为数据采集要求：固定时间采样（上午 10：00），固定时长（猕猴单笼自由活动 1 小时），固定距离（录像机位于笼子前方 1 米处），在没有外部干扰的情况下记录。
c. 病理数据采集要求：黑质多巴胺能神经元主要位于黑质致密部，我们在 4×物镜（3.5 mm × 2.6 mm）、20×物镜（690.8 μm × 518.1 μm）和40×物镜（345.4 μm × 259 μm）下鉴定具备典型多巴胺能神经元形态且呈现 TH 免疫组化阳性染色的细胞，以第三对脑神经 3n 为参考，选定黑质长轴近中心区域 40×物镜拍照用于细胞计数。
d. 以科学规范为原则，每条数据的采集、描述与数字化表达的每个环节均由专业人员操作，保证信息准确信及科学性。实验数据源于仪器测
量，逐一录入。

数据来源：a.行为学数据
为了评估PD症状，我们从两方面收集行为数据：1）记录食蟹猴的日常行为，量化PD症状，使用改进版Kurlan量表（旧大路猴帕金森病经典评定量表）进行评分；2）通过左旋多巴（L-dopa）或阿扑吗啡（Apo）诱导的症状改善进行评定。
我们利用数码相机（Sony HDR-XR260，日本）收集此例食蟹猴的录像。实验期间，共收集行为数据三次。
b.PD病理染色
TH染色：食蟹猴双侧黑质的冠状切片用3% H2O2（5 min）、3% triton X-100（4 min，中国北京索莱宝）和山羊血清（15 min，中国福州迈新）处理，然后兔抗TH抗体（1:1000，AB152，Millipore）4 °C过夜。次日，切片与二抗（PV-9000，30 min，中国北京中杉金桥）37 °C孵育。之后DAB显色2 min（DAB-1031 Kit，20×，中国福州迈新），苏木精复染细胞核。
PSer129αS染色：切片用10 μM/ mL蛋白酶K（中国上海阿拉丁）在10 mM Tris-HCl，pH=7.8、100 mM NaCl，0.1％ Tween-20（Bio-Rad，美国）中37°C处理30 min。之后用3％ H2O2（5 min，中国福州迈新）、3％ Triton X-100（4 min，中国北京索莱宝），10％山羊血清处理（15 min，中国福州迈新），经典的兔抗PSer129αS多克隆抗体（1：100，ab59264，Abcam，美国）4°C过夜。次日，切片与抗兔/小鼠二抗试剂盒（PV 9000，中国北京中杉金桥）37°C孵育30 min。之后DAB显色2 min（DAB-1031Kit，20×，中国福州迈新），苏木精复染细胞核。
染色后，所有切片乙醇梯度脱水并用二甲苯透明，中性树脂封片，光学显微镜（奥林匹斯，CX41；照相机：奥林匹斯DP25；软件：cellSens Entry 1.4.1；日本）采集照片。
数据生产方式：a.行为学数据
两名有经验的视频分析员参与录像分析。评分者不知此猴的实验操作，使用改进版Kurlan量表（Smith RD, Zhang Z, Kurlan R, McDermott M, Gash DM. Developing a stable bilateral model of parkinsonism in rhesus monkeys. Neuroscience 1993, 52: 7-16）对视频片段打分。该量表包括4个部分：A部分：帕金森病特征；B部分：药物副作用；C部分：总体活动水平；D部分：临床分期。PD评分依据A部分得出，总分为20，包括震颤：0-3；姿势：0-2；步态：0-4；运动迟缓：0-4；平衡：0-2；总体运动技能：0-3；防御反应：0-2。两位评分者的得分如果没有明显差异（少于两分），将数据汇总；若评分出现较大差异，则由实验者检查，最终评分将通过一起观看视频确定。在旋转计数中，两名视频分析员同样不知猕猴的实验操作，统计视频中猕猴的旋转次数。
b.PD病理特征染色数据
通过ImageJ软件（National Institutes of Health，Bethesda，Maryland，USA）在40×物镜下对选定区域的黑质多巴胺能神经元（TH阳性染色细胞）计数。分别算出黑质的平均细胞密度（个数/mm2）。为了验证计数方法的可靠性，黑质多巴胺能神经元的总数通过与Garcia等（Garcia-Dominguez I, Vesela K, Garcia-Revilla J, Carrillo-Jimenez A, Roca-Ceballos MA, Santiago M, et al. Peripheral Inflammation Enhances Microglia Response and Nigral Dopaminergic Cell Death in an in vivo MPTP Model of Parkinson's Disease. Front Cell Neurosci 2018, 12: 398）和Gundersen等（Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988, 96: 857-881）使用的方法估算，与报道的结果接近。黑质PSer129αS聚集计数时，发现阳性染色信号则进行统计。
时间范围：2016 年 03 月 01 日- 2021 年 03 月 20 日

We identified a 10-year-old male cynomolgus monkey (*Macaca fascicularis*, roughly equivalent to a 30–40-year-old human) with suspected spontaneously occurring Parkinson's disease (PD). The monkey exhibited typical clinical PD symptoms, including bradykinesia, resting tremor, and postural instability. The therapeutic effects of L-dopa and apomorphine were consistent with the responses observed in PD patients. Pathological staining revealed marked morphological damage and significant loss (~70%) of nigral dopaminergic neurons. Intracellular phosphorylated alpha-synuclein aggregates were increased, along with Lewy neurites, and there was prominent proliferation and activation of nigral microglia and astrocytes. Additionally, the cerebrospinal fluid (CSF) dopamine levels of this monkey were significantly lower than those of controls. Its pathogenesis may be associated with LRRK2 mutations.

By referring to the sequencing results of 11 PD risk genes from over 1,500 cynomolgus monkeys, we found rare missense mutations in the LRRK2 and ATP13A2 genes of this monkey. In vitro functional validation indicated that the LRRK2 mutation may promote PD pathogenesis (e.g., increased phosphorylated LRRK2 and phosphorylated Rab10; RNA-seq revealed activated pathways involving immune abnormalities and inflammatory responses).

This work is the first to confirm that, in addition to humans, monkeys can also develop spontaneous PD, with an age of onset mimicking the characteristics of early-onset PD in humans, and the LRRK2 mutation also reflects the potential "gain-of-function" pathogenesis of PD patients.

Data Quality:
a. Experimental Animals and Ethics
The experimental protocol and animal welfare were approved by the Institutional Animal Care and Use Committee (IACUC) of the Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC No. 20041). Macaques (including rhesus monkeys and cynomolgus monkeys) were housed individually in cages under a standard light/dark cycle, with free access to food and water, and cared for by veterinarians. A total of 60 adult monkeys were involved in the screening for spontaneous PD.

b. Behavioral Data Collection Requirements: Sampling was performed at a fixed time (10:00 AM), for a fixed duration (1 hour of free activity of the single-caged macaque), and at a fixed distance (video camera positioned 1 meter in front of the cage). Recordings were made without external interference.

c. Pathological Data Collection Requirements: Nigral dopaminergic neurons are primarily located in the substantia nigra pars compacta. We identified cells with typical dopaminergic neuron morphology and positive tyrosine hydroxylase (TH) immunohistochemical staining using 4× (3.5 mm × 2.6 mm), 20× (690.8 μm × 518.1 μm), and 40× (345.4 μm × 259 μm) objective lenses. Using the third cranial nerve (3n) as a reference, we selected the near-central region of the long axis of the substantia nigra for photographing at 40× magnification for cell counting.

d. Following the principle of scientific standardization, every step of data collection, description, and digital expression was operated by professional personnel to ensure accuracy and scientific validity. Experimental data were obtained through instrument measurements and entered one by one.

Data Sources:
a. Behavioral Data
To evaluate PD symptoms, we collected behavioral data from two aspects: 1) Recording the daily behaviors of the cynomolgus monkey and quantifying PD symptoms using a modified Kurlan scale (the classic Old World monkey Parkinson's disease rating scale); 2) Assessing symptom improvement induced by L-dopa or apomorphine (Apo). We used a digital camera (Sony HDR-XR260, Japan) to collect videos of this cynomolgus monkey. A total of three sets of behavioral data were collected during the experiment.

b. PD Pathological Staining
TH Staining: Coronal sections of the bilateral substantia nigra of the cynomolgus monkey were treated with 3% H2O2 (5 min), 3% Triton X-100 (4 min, Solarbio, Beijing, China), and goat serum (15 min, Maixin Bio, Fuzhou, China), then incubated with rabbit anti-TH antibody (1:1000, AB152, Millipore) overnight at 4 °C. The next day, sections were incubated with secondary antibody (PV-9000, 30 min, ZSGB-BIO, Beijing, China) at 37 °C. Subsequently, DAB staining was performed for 2 min (DAB-1031 Kit, 20×, Maixin Bio, Fuzhou, China), followed by nuclear counterstaining with hematoxylin.

PSer129αS Staining: Sections were treated with 10 μM/mL proteinase K (Aladdin, Shanghai, China) in 10 mM Tris-HCl, pH=7.8, 100 mM NaCl, 0.1% Tween-20 (Bio-Rad, USA) at 37 °C for 30 min. Then, sections were treated with 3% H2O2 (5 min, Maixin Bio, Fuzhou, China), 3% Triton X-100 (4 min, Solarbio, Beijing, China), and 10% goat serum (15 min, Maixin Bio, Fuzhou, China), followed by incubation with rabbit anti-PSer129αS polyclonal antibody (1:100, ab59264, Abcam, USA) overnight at 4 °C. The next day, sections were incubated with anti-rabbit/mouse secondary antibody kit (PV 9000, ZSGB-BIO, Beijing, China) at 37 °C for 30 min. Subsequently, DAB staining was performed for 2 min (DAB-1031 Kit, 20×, Maixin Bio, Fuzhou, China), followed by nuclear counterstaining with hematoxylin.

After staining, all sections were dehydrated in a graded ethanol series, cleared with xylene, mounted with neutral resin, and photographed using an optical microscope (Olympus, CX41; camera: Olympus DP25; software: cellSens Entry 1.4.1; Japan).

Data Generation Methods:
a. Behavioral Data
Two experienced video analysts participated in video analysis. The scorers were blinded to the experimental manipulation of the monkey, and scored video clips using the modified Kurlan scale (Smith RD, Zhang Z, Kurlan R, McDermott M, Gash DM. Developing a stable bilateral model of parkinsonism in rhesus monkeys. Neuroscience 1993, 52: 7-16). This scale includes four sections: Section A: Parkinson's disease characteristics; Section B: Drug side effects; Section C: Overall activity level; Section D: Clinical staging. PD scores were based on Section A, with a total score of 20, including tremor (0–3), posture (0–2), gait (0–4), bradykinesia (0–4), balance (0–2), overall motor skills (0–3), and defensive response (0–2). If the scores of the two analysts had no significant difference (less than 2 points), the data were pooled; if there was a large difference in scores, the experimenter would check and the final score was determined by jointly viewing the video. For rotation counting, the two video analysts were also blinded to the experimental manipulation of the macaque, and counted the number of rotations of the macaque in the video.

b. PD Pathological Feature Staining Data
The number of nigral dopaminergic neurons (TH-positive stained cells) in the selected region was counted at 40× magnification using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). The average cell density of the substantia nigra (number/mm²) was calculated separately. To verify the reliability of the counting method, the total number of nigral dopaminergic neurons was estimated using methods used by Garcia et al. (Garcia-Dominguez I, Vesela K, Garcia-Revilla J, Carrillo-Jimenez A, Roca-Ceballos MA, Santiago M, et al. Peripheral Inflammation Enhances Microglia Response and Nigral Dopaminergic Cell Death in an in vivo MPTP Model of Parkinson's Disease. Front Cell Neurosci 2018, 12: 398) and Gundersen et al. (Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988, 96: 857-881), and the results were consistent with reported values. When counting PSer129αS aggregates in the substantia nigra, positive staining signals were counted.

Time Range: March 1, 2016 – March 20, 2021