使用预突子形成测定使用预突子组织者珠和"神经元球"文化
Summary
预突生形成是一个动态过程,包括以适当的顺序积累突触蛋白。在这种方法中,前突子形成由与”神经元球”培养的正交片上与前突子组织者蛋白结合的珠子触发,因此在先发形成期间很容易分析突触蛋白的积累。
Abstract
在神经元发育过程中,突触形成是建立神经回路的重要一步。要形成突触,突触蛋白必须通过从细胞体的传输和/或未成熟的突触中的局部翻译以适当的顺序提供。然而,它不完全了解突触蛋白如何积累在突触以适当的顺序。本文提出了一种利用神经元球培养与珠子结合来诱导前突触形成的新方法。神经元细胞聚集的神经元球提供远离细胞体和树突的斧状片,因此可以通过避免细胞体的压倒性信号来检测前突子的弱荧光信号。作为珠子来触发前突动的形成,我们使用珠子与富含亮氨酸的重复跨膜神经元2(LRRTM2)结合,这是一个兴奋的先发前的组织者。使用这种方法,我们证明了囊性谷氨酸输送剂1(vGlut1),一种突触性囊泡蛋白,在预突触中积累得比Munc18-1(一种活性区蛋白)快。Munc18-1 在切除细胞体后,在先行中积累翻译依赖性。这一发现表明Munc18-1通过局部平移在斧子中积累,而不是从细胞体传输。总之,该方法适用于分析突触前蛋白的积累和突触蛋白的来源。由于神经元球培养简单,无需使用特殊仪器,该方法可适用于其他实验平台。
Introduction
突触形成是神经回路1、2、3开发过程中的关键步骤之一。由突触前和突触后组成的专门隔间的形成是一个复杂和多步骤的过程,涉及适当识别斧子和树突,形成活动区和后突密度,以及正确对齐电位通道和神经递质受体1,2。在每个过程中,许多种类的突触蛋白通过从细胞体中运输突触蛋白和/或通过隔间中的局部翻译,在适当的时间积累到这些专用隔间。这些突触蛋白被认为是以有组织的方式排列,以形成功能性突触。一些突触蛋白的机能导致神经疾病4,5。然而,目前还不清楚突触蛋白是如何在适当的时间积累。
为了研究突触蛋白如何以有组织的方式积累,有必要按时间顺序检查突触蛋白的积累。一些报告演示了实时成像,以观察神经元分离培养的突触形成6,7。然而,它是费时的神经元,刚刚开始在显微镜下突触形成。为了有效地观察突触蛋白的积累,突触的形成必须在研究人员想要诱导形成的时候开始。第二个挑战是区分突触蛋白的积累,由于从细胞体或突触中的局部翻译。为此,翻译水平是必要的,在不允许从细胞体运输突触蛋白的条件下进行测量。
我们利用神经元球培养物与珠子的结合,开发出了新型的先发性形成测定,以诱导前突子形成8。神经元球培养是开发来检查斧状表型,由于形成围绕细胞体的斧板9,10。我们使用磁珠与富含亮氨酸的重复跨膜神经元2(LRRTM2)结合,这是一个前突触组织者诱导兴奋前突触(图1A)11,12,13。通过使用 LRRTM2 磁珠,在应用磁珠时开始预突子形成。这意味着,前突子的形成开始于神经元球的千个斧子在同一时间,因此它允许有效地检查突触蛋白积累的精确时间过程。此外,神经元球培养很容易通过去除细胞体来阻止从体中传递突触蛋白(图1B)8。我们已经证实,没有细胞体的斧子可以存活,并在去除细胞体后至少4小时健康。因此,此方案适用于研究突触蛋白的衍生位置(细胞体或斧子),以及突触蛋白如何以有组织的方式积累。
Protocol
Representative Results
Discussion
我们开发了利用神经元球培养法检测用LRRTM2-珠刺激的先增前形成的方法。目前,大多数前突生形成测定包括多-D-流源(PDL)涂层珠和分离培养/微流体室20,21,22。该方法的优点之一是LRRTM2珠子。当LRRTM2与神经素相互作用,形成兴奋的先阴突子,特别是11,12,13,PDL-珠诱导兴奋和抑制预突生非<…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到JSPS科学研究援助赠款(C)(第22500336号、25430068号、16K07061号)(Y. Sasaki)的支持。我们感谢野中泰郎博士和木崎美子女士(横滨城市大学)提供生物素素的LRRTM2蛋白。我们还感谢小池和石井的技术援助。
Materials
| Antibody diluent | DAKO | S2022 | |
| Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-585-151 | |
| Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-545-152 | |
| mouse anti-Munc18-1 | BD Biosciences | 610336 | |
| B-27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504044 | |
| Bovine Serum Alubumin (BSA) | Nacalai Tesque | 01863-48 | |
| Cell-Culture Treated Multidishes (4 well dish) | Nunc | 176740 | |
| Complete EDTA-free | Roche | 11873580001 | |
| cooled CCD camera | Andor Technology | iXON3 | |
| Coverslip | Matsunami | C015001 | Size: 15 mm, Thickness: 0.13-0.17 mm |
| Cytosine β-D-arabinofuranoside (AraC) | Sigma-Aldrich | C1768 | |
| 4',6-Diamidino-2-phenylindole Dihydrochloride (DAPI) | Nacalai Tesque | 11034-56 | |
| Deoxyribonuclease 1 (DNase I) | Wako pure chemicals | 047-26771 | |
| Expi293 Expression System | Thermo Fisher Scientific | A14635 | |
| Horse serum | Sigma-Aldrich | H1270 | |
| image acquisition software | Nikon | NIS-element AR | |
| Image analysis software | NIH | Image J | https://imagej.nih.gov/ij/ |
| Inverted fluorecent microscope | Nikon | Eclipse Ti-E | |
| GlutaMAX | Thermo Fisher Scientific | 35050061 | |
| Neurobasal media | Thermo Fisher Scientific | #21103-049 | |
| Normal Goat Serum (NGS) | Thermo Fisher Scientific | #143-06561 | |
| N-propyl gallate | Nacalai Tesque | 29303-92 | |
| Paraformaldehyde (PFA) | Nacalai Tesque | 26126-25 | |
Paraplast Plus |
Sigma-Aldrich | P3558 | |
| Poly-L-lysine Hydrobromide (MW > 300,000) | Nacalai Tesque | 28359-54 | |
| poly (vinyl alcohol) | Sigma | P8136 | |
| Prepacked Disposable PD-10 Columns | GE healthcare | 17085101 | |
| rabbit anti-vesicular glutamate transporter 1 | Synaptic Systems | 135-302 | |
| SCAT 20X-N (neutral non-phosphorous detergent) | Nacalai Tesque | 41506-04 | |
| Streptavidin-coated magnetic particles | Spherotech Inc | SVM-40-10 | diameter: 4-5 µm |
| TritonX-100 | Nacalai Tesque | 35501-15 | |
| Trypsin | Nacalai Tesque | 18172-94 |
Referências
- Waites, C. L., Craig, A. M., Garner, C. C. Mechanisms of Vertebrate Synaptogenesis. Annual Review of Neuroscience. 28 (1), 251-274 (2005).
- Ziv, N. E., Garner, C. C. Cellular and molecular mechanisms of presynaptic assembly. Nature Reviews Neuroscience. 5 (5), 385-399 (2004).
- Chia, P. H., Li, P., Shen, K. Cellular and molecular mechanisms underlying presynapse formation. Journal of Cell Biology. 203 (1), 11-22 (2013).
- Südhof, T. C. Neuroligins and neurexins link synaptic function to cognitive disease. Nature. 455 (7215), 903-911 (2008).
- Saitsu, H., et al. CASK aberrations in male patients with Ohtahara syndrome and cerebellar hypoplasia. Epilepsia. 53 (8), 1441-1449 (2012).
- Friedman, H. V., Bresler, T., Garner, C. C., Ziv, N. E. Assembly of New Individual Excitatory Synapses. Neuron. 27 (1), 57-69 (2000).
- Bresler, T., et al. Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. The Journal of neuroscience the official journal of the Society for Neuroscience. 24 (6), 1507-1520 (2004).
- Parvin, S., Takeda, R., Sugiura, Y., Neyazaki, M., Nogi, T., Sasaki, Y. Fragile X mental retardation protein regulates accumulation of the active zone protein Munc18-1 in presynapses via local translation in axons during synaptogenesis. Neuroscience Research. , (2018).
- Sasaki, Y., et al. Phosphorylation of Zipcode Binding Protein 1 Is Required for Brain-Derived Neurotrophic Factor Signaling of Local β-Actin Synthesis and Growth Cone Turning. Journal of Neuroscience. 30 (28), 9349-9358 (2010).
- Sasaki, Y., Gross, C., Xing, L., Goshima, Y., Bassell, G. J. Identification of axon-enriched microRNAs localized to growth cones of cortical neurons. Developmental neurobiology. 74 (3), 397-406 (2014).
- Linhoff, M. W., et al. An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers. Neuron. 61 (5), 734-749 (2009).
- de Wit, J., et al. LRRTM2 Interacts with Neurexin1 and Regulates Excitatory Synapse Formation. Neuron. 64 (6), 799-806 (2009).
- Ko, J., Fuccillo, M. V., Malenka, R. C., Südhof, T. C. LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation. Neuron. 64 (6), 791-798 (2009).
- Kaech, S., Banker, G. Culturing hippocampal neurons. Nature protocols. 1 (5), 2406-2415 (2006).
- Hirai, H., et al. Structural basis for ligand capture and release by the endocytic receptor ApoER2. EMBO reports. , (2017).
- Yasui, N., et al. Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region. Journal of Biological Chemistry. 286 (40), 35247-35256 (2011).
- Bassell, G. J., Warren, S. T. Fragile X Syndrome: Loss of Local mRNA Regulation Alters Synaptic Development and Function. Neuron. 60 (2), 201-214 (2008).
- Darnell, J. C., Klann, E. The translation of translational control by FMRP: therapeutic targets for FXS. Nat Neurosci. 16 (11), 1530-1536 (2013).
- Richter, J. D., Bassell, G. J., Klann, E. Dysregulation and restoration of translational homeostasis in fragile X syndrome. Nature Reviews Neuroscience. 16 (10), 595-605 (2015).
- Lucido, A. L., et al. Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts. Journal of Neuroscience. 29 (40), 12449-12466 (2009).
- Taylor, A. M., Wu, J., Tai, H. C., Schuman, E. M. Axonal Translation of β-Catenin Regulates Synaptic Vesicle Dynamics. Journal of Neuroscience. 33 (13), 5584-5589 (2013).
- Batista, A. F. R., Martínez, J. C., Hengst, U. Intra-axonal Synthesis of SNAP25 Is Required for the Formation of Presynaptic Terminals. Cell reports. 20 (13), 3085-3098 (2017).
