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：
10003731
负责人：
Huaibin Cai
金额：
$ 2.6万
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/10003731
关键词：
ALS2 gene 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 Impairment Induced Mutation Interneurons Knockout Mice Knowledge Lead Length Location Longevity Lumbar spinal cord structure Mediating Membrane Proteins Messenger RNA Missense Mutation Molecular Monitor Motor Motor Activity Motor Neuron Disease Motor Neurons Movement Mus Muscarinic M2 Receptor Mutation N-terminal Neurons Pathogenicity Pathologic Pathway interactions Pattern Phenotype Physiological Presynaptic Terminals Property Proteins Reporting Research Resources Rest Rotarod Performance Test Site Solubility Spinal Spinal Cord Stains Structure Synapses Synaptophysin Syndrome System Systems Analysis Tertiary Protein Structure 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 field study gain of function gait examination immunoreactivity in vivo male motor control motor disorder motor impairment motor neuron degeneration mouse model mutant nerve supply neural circuit neurodevelopment neuronal cell body novel overexpression 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种独立的WT（A6，B3和B7）和P56（C8，C11和D3）VAPB转基因（TG）小鼠的独立线。与同窝非Transmate非Transgenic（NTG）相比控件。然而，与WT VAPB蛋白（20倍相对于内源性相比内源性），p56s VAPB蛋白的稳定水平明显少得多，这表明p56S突变可能会损害VAPB的稳定性和溶解度。体内蛋白质。后来，我们将这两种最高的VAPB表达线称为下游行为和病理研究中的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蛋白由脊柱运动神经元高度表达（图1D），并且在皮质脊髓运动神经元中也很丰富。 WT VAPB的过表达导致WT VAPB TG小鼠神经元中VAPB蛋白的弥漫性胞质分布模式。相比之下，在p56S vapb TG小鼠的神经元中观察到了VAPB的大型点状染色。 p56s VAPB蛋白的明显异常聚类可能会影响神经元的功能和存活。 2。P56SVAPB TG小鼠发展出异常运动行为表型密切监测了39只雄性小鼠的队列（NTG为15只小鼠，wt 12，P56S VAPB的12个）的队列被密切监测用于运动和其他行为表型。 p56s和WT VAPB TG小鼠均可生存，正常发育，并且寿命正常。然而，与同窝NTG和WT VAPB小鼠相比，p56s VAPB小鼠体重的增加显着少得多。在12个月大时，p56s VAPB小鼠意外地开始在开放式测试中在水平和垂直运动中表现出显着的多动症。此外，Rotarod测试显示，p56S VAPB小鼠的运动协调/平衡显着损害，也从12个月大时开始。由于步态异常是运动神经元变性的其他小鼠模型中运动功能障碍的早期迹象，因此我们使用TREADSCAN步态分析系统对P56S VAPB小鼠的步态参数进行了量化。有趣的是，与同窝NTG对照组和WT TG小鼠相比，p56s VAPB小鼠的步幅长度和步步时间都显着较短。 p56s VAPB TG小鼠的较短步伐在两个月大时才被检测到，并且在其余生中没有明显恶化。在P56S VAPB TG小鼠的C11线中也观察到了类似的步幅长度。但是，显示出正常的运动活动。相比之下，WT VPAB和对照NTG小鼠之间的步幅长度没有显着差异。 p56s VAPB小鼠中步态参数的早期发作和非进行性改变表明p56s VAPB可能会影响调节小鼠步态特性的神经回路的发展。 3。P56SVAPB TG小鼠发展了皮质脊髓运动神经元的进行性变性鉴于VAPB中的p56S突变会导致运动神经元变性，因此发现p56S VAPB TG小鼠出现了渐进的多动症，这似乎是违反直觉的。但是，先前的报告表明，缺乏皮质脊髓运动神经元的FEZ样（FEZL）敲除小鼠也表现出明显的多动症。这种现象可以归因于人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小鼠的过度活跃性的病理基础，我们检查了p56s VAPB TG小鼠的脑脊髓皮层神经元的生存能力。可以通过对转录因子政变TF1相互作用蛋白2（CTIP2）进行免疫染色，可以轻松地在大脑皮层内列内鉴定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，在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降低（图3）。除皮层脊髓运动神经元的丧失外，还主要发现GFAP阳性反应性星形胶质细胞显着增加，主要在18个月大的P56S小鼠的大脑皮层中。明显的是，在C11线p56s p56s p56S vapb TG小鼠的大脑皮层中，没有明显的皮质脊髓运动神经元或反应性神经胶质变性，这在皮质中显示突变体VAPB的较低水平表达，并且仅表现出步态异常。总之，我们记录了老年p56S VAPB TG小鼠中皮质脊髓运动神经元的选择性丧失，这可能有助于这些突变动物中发生的进行性多动症。 4。在老年p56s VAPB TG小鼠中没有明显的脊柱运动神经元变性为了研究VAPB中的p56S突变是否导致脊柱运动神经元变性，我们在18个月大的p56s和WT VAPB TG小鼠以及NTG的同胞对照组中计算了运动神经元。 p56s VAPB TG小鼠（11.65 +/- 0.62）中每个部分的脊柱运动神经元数与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小鼠的脊髓中反应性神经胶质的发生。但是，我们没有观察到p56s VAPB TG小鼠的脊髓中反应性神经胶质的显着增加。 5。P56S突变诱导了VAPB蛋白在脊柱运动神经元中C-Bouton的突触后位点的转运与我们在皮质脊髓运动神经元中的观察结果相反，在p56s vapb小鼠的脊柱运动神经元中，没有观察到在VAPB阳性大点点结构中钙钙蛋白钙钙蛋白钙钙蛋白网虫产生的明显共染色。取而代之的是，我们发现一小部分的VAPB免疫反应性与p56S vapb TG小鼠的脊柱运动神经元中的突触蛋白（SYN）并列时，当我们与SYN（SYN）共染色时，SYN（一种标记）是前端端的标记。我们进一步证实了用VAPB抗体免疫-EM通过免疫EM证实了VAPB蛋白在p56S VAPB小鼠的脊柱运动神经元中的突触后位置。作为对照，在WT VAPB TG和对照NTG小鼠的脊柱运动神经元中未发现VAPB的突触后位置。突变体VAPB相关的突触较大，仅限于脊柱运动神经元的SOMA和近端树突，属于特殊的突触，即脊柱运动神经元的C-Bouton。 C-Boutons从中央脊髓中央管附近的一组胆碱能中间神经元中接受胆碱能神经。沿大脊柱运动神经元的质膜分布的2型毒蕈碱（M2）受体均匀分布，介导C-Boutons的突触后反应，并调节脊柱运动神经元的兴奋性。因此，我们发现VAPB的免疫反应性与p56s VAPB小鼠的脊柱运动神经元中的胆碱乙酰转移酶（CHAT）并置。此外，VAPB蛋白在脊柱运动神经元的细胞表面显示了特异性的共定位，表明p56s VAPB的功能上的潜在增益在影响脊柱运动神经元中C-Boutons的正常结构和功能方面的正常结构和功能。