Function and Pathogenic Mechanism of VAPB in ALS and Other Motor Neuron Diseases

VAPB在ALS及其他运动神经元疾病中的作用及发病机制

基本信息

批准号：
9147383
负责人：
Huaibin Cai
金额：
$ 8.83万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
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/9147383
关键词：
Affect Age Age-Months Animals Antibodies Astrocytes Behavioral Biological Assay Brain Calnexin Cell membrane Cell surface Cells Central cord canal structure Cerebral cortex Choline O-Acetyltransferase Critical Pathways DNA Sequence Alteration Data Dendrites Deterioration Development Diffuse Endoplasmic Reticulum Enzymes Equilibrium Exhibits Face Family Free Radical Scavenging Future Gait Gait abnormality Generations Genes Genetic Glial Fibrillary Acidic Protein Gliosis Golgi Apparatus Human Hyperactive behavior Immunohistochemistry Induced Mutation Interneurons Knockout Mice Knowledge Lead Length Life Location Longevity Lumbar spinal cord structure Mediating Membrane Messenger RNA Missense Mutation Molecular Monitor Motor Motor Activity Motor Neuron Disease Motor Neurons Movement Mus Muscarinic M2 Receptor Mutation N-terminal Neurons Pathway interactions Pattern Phenotype Physiological Presynaptic Terminals Property Proteins Reporting Research Resources Rest Rotarod Performance Test Site Solubility Spinal Spinal Cord Staining method Stains Structure Synapses Synaptophysin Syndrome System Tertiary Protein Structure Testing Time Transgenic Mice Transgenic Organisms Weight Gain aged base biological adaptation to stress chicken ovalbumin upstream promoter-transcription factor cholinergic cohort dynactin early onset endoplasmic reticulum stress gain of function gait examination immunoreactivity in vivo interest male motor control motor disorder motor impairment motor neuron degeneration mouse model mutant nerve supply neural circuit neuronal cell body novel postsynaptic protein B protein TDP-43 response sperm protein superoxide dismutase 1 vesicle-associated membrane protein

项目摘要

1. Generation of human wild-type and P56S VAPB transgenic mice We have obtained three independent lines of WT (A6, B3, and B7) and P56S (C8, C11, and D3) VAPB transgenic (Tg) mice, respectively. More than a 20-fold over-expression of VAPB mRNA was observed in the brain of line B3 of WT (25.6 1.9 fold) and line D3 of P56S VAPB Tg mice (23.7 0.7 fold) as compared to littermate non-transgenic (nTg) controls. However, the steady level of P56S VAPB protein (7-fold vs. endogenous) was significantly less compared to WT VAPB protein (20-fold vs. endogenous) in whole brain lysate, indicating that the P56S mutation might impair stability and solubility of VAPB protein in vivo. We later refer to these two highest VAPB expression lines as WT and P56S VAPB Tg mice in the downstream behavioral and pathological studies. To examine the cellular and subcellular distribution of VAPB, we performed immunohistochemistry with a VAPB antibody, and found that endogenous VAPB protein is highly expressed by spinal motor neurons (Fig. 1D) and is also abundant in corticospinal motor neurons. Over-expression of WT VAPB led to a diffuse cytosolic distribution pattern of VAPB protein in neurons of WT VAPB Tg mice. By contrast, widespread large punctate staining of VAPB was observed in neurons of P56S VAPB Tg mice. This apparent abnormal clustering of P56S VAPB protein may potentially affect the function and survival of neurons. 2. P56S VAPB Tg mice developed abnormal motor behavioral phenotypes A cohort of 39 male mice (15 for nTg, 12 for WT, and 12 for P56S VAPB) were closely monitored for motor and other behavioral phenotypes. Both P56S and WT VAPB Tg mice were viable, developed normally, and lived a normal life span. However, the gain of body weight of P56S VAPB mice was significantly less compared to littermate nTg and WT VAPB mice after 15 months of age. P56S VAPB mice unexpectedly started to exhibit significant hyperactivity in both horizontal and vertical movements in the Open-field test at 12 months of age. In addition, Rotarod test revealed a significant impairment of motor coordination/balance in P56S VAPB mice beginning at 12 months of age, too. Since gait abnormalities are an early sign of motor dysfunction in other mouse models of motor neuron degeneration, we quantified the gait parameters of P56S VAPB mice using the Treadscan Gait Analysis system. Interestingly, both the stride length and stride time of P56S VAPB mice were significantly shorter compared to littermate nTg controls and WT Tg mice. The shorter stride of P56S VAPB Tg mice was first detected at two months of age and was persistent without significant deterioration through the rest of their lives. A similar shortening of stride length was also observed in line C11 of P56S VAPB Tg mice; which however, displayed normal locomotor activities. By contrast, no significant difference in the stride length was found between WT VPAB and control nTg mice. The early onset and non-progressive alteration of gait parameters in P56S VAPB mice indicates P56S VAPB may affect the development of neural circuitry regulating the gait properties of mice. 3. P56S VAPB Tg mice developed progressive degeneration of corticospinal motor neurons Given that the P56S mutation in VAPB causes motor neuron degeneration, it seems counterintuitive to discover that P56S VAPB Tg mice developed a progressive hyperactivity. However, a previous report shows that Fez-like (Fezl) knockout mice, which lack the corticospinal motor neurons, also exhibit significant hyperactivity. This phenomenon could be attributed to different wiring or properties of corticospinal motor neurons in human and mouse motor control systems. To explore the pathological basis of hyperactivity in aged P56S VAPB Tg mice, we examined the viability of corticospinal motor neurons in the cerebral cortex of P56S VAPB Tg mice. The corticospinal motor neurons can be easily identified within layerV of the cerebral cortex by immunostaining with an antibody against transcription factor COUP TF1-interacting protein 2 (CTIP2). We found a significant reduction of CTIP2-positive corticospinal motor neurons in the cortex of 18-month old P56S VAPB Tg mice as compared to age-matched WT and control nTg mice (Fig. 3). In addition to the loss of corticospinal motor neurons, a significant increase of GFAP-positive reactive astrocytes was also found mainly in the cerebral cortex of 18-month old P56S mice. Noticeably, no significant degeneration of corticospinal motor neurons or reactive gliosis was detected in the cerebral cortex of C11 line P56S VAPB Tg mice, which showed lower level expression of mutant VAPB in the cortex and only exhibited gait abnormalities. Together, we document a rather selective loss of corticospinal motor neurons in aged P56S VAPB Tg mice, which may contribute to the progressive hyperactivity developed in these mutant animals. 4. No significant degeneration of spinal motor neurons in aged P56S VAPB Tg mice To investigate whether the P56S mutation in VAPB leads to spinal motor neuron degeneration, we counted motor neurons in the lumbar spinal cord of 18-month old P56S and WT VAPB Tg mice as well as nTg littermate controls. The number of spinal motor neurons per section in P56S VAPB Tg mice (11.65 +/- 0.62) was comparable with those in WT VAPB (12.15  0.57, p=0.8) and control nTg mice (13.01+/- 0.77, p=0.6). We also examined the occurrence of reactive gliosis in the spinal cord of 18 month-old P56S VAPB Tg mice. However, we did not observe any significant increase of reactive gliosis in the spinal cord of P56S VAPB Tg mice. 5. The P56S mutation induced a translocation of VAPB protein to the postsynaptic site of C-boutons in spinal motor neurons In contrast to our observations in corticospinal motor neurons, no apparent co-staining of calnexin in VAPB-positive large punctuate structures was observed in the spinal motor neurons of P56S VAPB mice. Instead, we found a small fraction of VAPB-immunoreactivity was juxtaposed with the synaptophysin (SYN) staining in the spinal motor neuron of P56S VAPB Tg mice when we co-stained VAPB with SYN, a marker for presynaptic terminals. We further confirmed the postsynaptic location of the VAPB protein in the spinal motor neuron of P56S VAPB mice by immuno-EM with a VAPB antibody. As controls, no postsynaptic location of VAPB was found in the spinal motor neuron of WT VAPB Tg and control nTg mice. The mutant VAPB-associated synapses were large and restricted to soma and proximal dendrites of spinal motor neurons, which belongs to a special class of synapses, the C-bouton of spinal motor neurons. C-boutons receive cholinergic innervation from a group of cholinergic interneurons near the central canal of spinal cord. The type 2 muscarinic (M2) receptors evenly distributed along the plasma membrane of large spinal motor neurons mediate the postsynaptic response of C-boutons and modulate the excitability of spinal motor neurons. Accordingly, we found the immunoreactivity of VAPB was juxtaposed with choline acetyltransferase (CHAT) staining in the spinal motor neuron of P56S VAPB mice. Furthermore, VAPB protein showed specific co-localization with M2 receptors at the cell surface of spinal motor neurons, indicating a potential gain of function of P56S VAPB in affecting the normal structure and function of C-boutons in spinal motor neurons.

1. 人类野生型和 P56S VAPB 转基因小鼠的产生我们分别获得了三个独立的WT（A6、B3和B7）系和P56S（C8、C11和D3）VAPB转基因（Tg）小鼠系。与同窝非转基因 (nTg) 小鼠相比，在 WT 系 B3 小鼠（25.6±1.9 倍）和 P56S VAPB Tg 小鼠系 D3（23.7±0.7 倍）的大脑中观察到 VAPB mRNA 过度表达超过 20 倍控制。然而，全脑裂解液中 P56S VAPB 蛋白的稳定水平（相对于内源性的 7 倍）明显低于 WT VAPB 蛋白（相对于内源性的 20 倍），表明 P56S 突变可能会损害 VAPB 的稳定性和溶解度体内的蛋白质。我们随后在下游行为和病理学研究中将这两个 VAPB 表达最高的系称为 WT 和 P56S VAPB Tg 小鼠。为了检查 VAPB 的细胞和亚细胞分布，我们用 VAPB 抗体进行了免疫组织化学，发现内源性 VAPB 蛋白在脊髓运动神经元中高度表达（图 1D），并且在皮质脊髓运动神经元中也丰富。 WT VAPB 的过度表达导致 WT VAPB Tg 小鼠神经元中 VAPB 蛋白的弥散性胞质分布模式。相比之下，在 P56S VAPB Tg 小鼠的神经元中观察到广泛的 VAPB 大点状染色。 P56S VAPB 蛋白的这种明显异常聚集可能会影响神经元的功能和存活。 2. P56S VAPB Tg 小鼠出现异常运动行为表型一组 39 只雄性小鼠（15 只为 nTg，12 只为 WT，12 只为 P56S VAPB）被密切监测运动和其他行为表型。 P56S 和 WT VAPB Tg 小鼠均可存活，发育正常，并具有正常寿命。然而，15月龄后，P56S VAPB小鼠的体重增加明显低于同窝nTg和WT VAPB小鼠。 P56S VAPB 小鼠在 12 个月大的旷场测试中出乎意料地开始在水平和垂直运动方面表现出明显的过度活跃。此外，Rotarod 测试显示，P56S VAPB 小鼠从 12 个月大起，运动协调/平衡也出现显着损害。由于步态异常是其他运动神经元变性小鼠模型运动功能障碍的早期征兆，因此我们使用 Treadscan 步态分析系统量化了 P56S VAPB 小鼠的步态参数。有趣的是，与同窝 nTg 对照和 WT Tg 小鼠相比，P56S VAPB 小鼠的步幅长度和步幅时间均显着缩短。 P56S VAPB Tg 小鼠的步幅较短是在两个月大时首次检测到的，并且在其余生中持续存在，没有明显恶化。在 P56S VAPB Tg 小鼠的 C11 系中也观察到类似的步幅缩短；然而，它表现出正常的运动活动。相比之下，WT VPAB 和对照 nTg 小鼠之间的步长没有显着差异。 P56S VAPB 小鼠步态参数的早期发生和非进行性改变表明 P56S VAPB 可能影响调节小鼠步态特性的神经回路的发育。 3. P56S VAPB Tg 小鼠皮质脊髓运动神经元进行性变性鉴于 VAPB 中的 P56S 突变会导致运动神经元变性，发现 P56S VAPB Tg 小鼠出现进行性多动似乎是违反直觉的。然而，之前的一份报告显示，缺乏皮质脊髓运动神经元的 Fez 样（Fezl）基因敲除小鼠也表现出明显的多动症。这种现象可能归因于人类和小鼠运动控制系统中皮质脊髓运动神经元的不同接线或特性。为了探索老年 P56S VAPB Tg 小鼠多动的病理基础，我们检查了 P56S VAPB Tg 小鼠大脑皮层皮质脊髓运动神经元的活力。通过使用针对转录因子 COUP TF1 相互作用蛋白 2 (CTIP2) 的抗体进行免疫染色，可以轻松识别大脑皮层 V 层内的皮质脊髓运动神经元。我们发现，与年龄匹配的 WT 和对照 nTg 小鼠相比，18 个月大的 P56S VAPB Tg 小鼠皮质中 CTIP2 阳性皮质脊髓运动神经元显着减少（图 3）。除了皮质脊髓运动神经元的损失外，还在 18 个月大的 P56S 小鼠的大脑皮层中发现了 GFAP 阳性反应性星形胶质细胞的显着增加。值得注意的是，在 C11 系 P56S VAPB Tg 小鼠的大脑皮层中没有检测到明显的皮质脊髓运动神经元变性或反应性神经胶质增生，其皮层中突变 VAPB 的表达水平较低，仅表现出步态异常。我们共同记录了老年 P56S VAPB Tg 小鼠皮质脊髓运动神经元的选择性丧失，这可能导致这些突变动物出现进行性多动症。 4.老年P56S VAPB Tg小鼠脊髓运动神经元无明显变性为了研究 VAPB 中的 P56S 突变是否导致脊髓运动神经元变性，我们对 18 个月大的 P56S 和 WT VAPB Tg 小鼠以及 nTg 同窝对照小鼠的腰脊髓中的运动神经元进行了计数。 P56S VAPB Tg 小鼠中每个切片的脊髓运动神经元数量 (11.65 +/- 0.62) 与 WT VAPB (12.15 ± 0.57, p=0.8) 和对照 nTg 小鼠 (13.01+/- 0.77, p=0.6) 相当。）。我们还检查了 18 个月大的 P56S VAPB Tg 小鼠脊髓中反应性神经胶质增生的发生情况。然而，我们没有观察到 P56S VAPB Tg 小鼠脊髓中反应性神经胶质增生的任何显着增加。 5. P56S突变诱导VAPB蛋白易位至脊髓运动神经元C-boutons的突触后位点与我们在皮质脊髓运动神经元中的观察相反，在 P56S VAPB 小鼠的脊髓运动神经元中没有观察到 VAPB 阳性大点状结构中钙连接蛋白的明显共染色。相反，当我们将 VAPB 与突触前末梢标记物 SYN 共染色时，我们发现 P56S VAPB Tg 小鼠脊髓运动神经元中一小部分 VAPB 免疫反应性与突触素 (SYN) 染色并列。我们通过使用 VAPB 抗体进行免疫电镜进一步证实了 P56S VAPB 小鼠脊髓运动神经元中 VAPB 蛋白的突触后位置。作为对照，在 WT VAPB Tg 和对照 nTg 小鼠的脊髓运动神经元中没有发现 VAPB 的突触后位置。突变的 VAPB 相关突触很大，仅限于脊髓运动神经元的体细胞和近端树突，属于一类特殊的突触，即脊髓运动神经元的 C-bouton。 C-boutons 接受来自脊髓中央管附近的一组胆碱能中间神经元的胆碱能神经支配。 2 型毒蕈碱 (M2) 受体沿着大脊髓运动神经元的质膜均匀分布，介导 C-boutons 的突触后反应并调节脊髓运动神经元的兴奋性。因此，我们发现 P56S VAPB 小鼠脊髓运动神经元中 VAPB 的免疫反应性与胆碱乙酰转移酶 (CHAT) 染色并列。此外，VAPB蛋白与脊髓运动神经元细胞表面的M2受体特异性共定位，表明P56S VAPB在影响脊髓运动神经元C-boutons的正常结构和功能方面具有潜在的功能增益。