一次繊毛を研究するために一次ニューロスフェア培養物を使用して
Summary
主な繊毛は、神経前駆細胞の増殖、ニューロン分化、および成人の神経機能における基本的に重要です。ここでは、繊毛形成とプライマリニューロスフェア培養を用いて神経幹/前駆細胞および分化したニューロンにおける繊毛へのシグナル伝達タンパク質の輸送を研究するための方法について説明します。
Abstract
The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.
Introduction
一次繊毛は、成体神経機能3における胚神経発達1、2、及び区画細胞内シグナル伝達の間ソニックヘッジホッグ（Shhの）経路を含む細胞シグナル伝達経路の調整における感覚アンテナとして機能する微小管ベースの動的細胞内区画である4 。このような5にパッチを当てShhの受容体として、これらの経路のシグナル伝達成分、。経路アクチベーターあるSmoothened（SMO）6。そしてGpr161 7、負のShh経路を調節するオーファンGタンパク質共役型受容体（GPCR）は、動的に繊毛に局在します。複数のGPCRは、10脳7、8、9で神経細胞に繊毛に局在することが報告されています</SUP>、11、12、13、14、15、16。繊毛および繊毛で生成シグナル伝達経路における欠陥は、複数の組織に影響を与えると総称ciliopathies 17、18、19として知られています。繊毛病の疾患スペクトルはしばしば、頭蓋顔面異常20、21、22のような神経発達の欠陥を含みます。加えて、視床下部ニューロンにおける主要繊毛は、中央満腹経路を調節し、欠陥は、バルデーBiedel症候群24として症候群ciliopathiesにおける肥満をミラーリング、中心性肥満23をもたらします。また、繊毛シグナリング神経ペプチド受容体はCENTRを調節しますアル満腹14、11経路。毛様体アデニリルシクラーゼIII（ACIII）の局在化および海馬ニューロンにおけるそのようなソマトスタチン受容体3としてGPCRは、新規物体認識欠陥および記憶欠損25をもたらし26と毛様体の整合27の欠如に匹敵します。繊毛が生成したシグナルの発達的側面は密接に組織恒常性に結びついています。特に、繊毛は小脳28、29における顆粒前駆細胞から生じるのShhサブタイプの髄芽腫の進行に重要です。このように、主繊毛は、胚、出生後、および成人の神経細胞の発達と機能の間に重要な役割を果たしています。
神経幹細胞（NSCは）、側脳室の脳室下帯（SVZ）に存在する海馬の歯状回の顆粒下ゾーン、及び哺乳類30、31、32における視床下部における第三脳室の脳室帯。 NSCは、多能ある自己複製能を有し、かつ脳の発達と再生医療30のために重要です。 SVZのほとんどのNSCは、静止していると、多くの場合、側脳室33に張り出し孤独主要繊毛を持っています。特にShhは、TGFβ、および受容体チロシンキナーゼ経路2、34、35、36に関連して、下流の細胞応答を誘導する様々な受容体の局在化を介して、一次繊毛信号。一次繊毛は、側脳室中に延びているので、一次繊毛は、脳脊髄液（CSF）のNSC 37を活性化するためにサイトカインを検出すると仮定されます</s>アップ。最近の研究では、Shhのシグナル伝達経路と主な繊毛が嗅上皮、肺、腎臓および38を含む複数の組織の修復および再生における幹細胞の活性化のために重要であることを示唆している39、40、41。しかしながら、CSFは、NSCを用いて一次繊毛が関与しているかどうかを通信するメカニズムは知られていません。文化の中で付着NSCは繊毛されています。このような繊毛でのSmoとGpr161としてのShh経路成分を、ローカライズ。そして42応答のShhです。このように、NSCは、Shhの経路を研究するための重要なモデル系、毛様体人身売買、および神経分化経路としての役割を果たすことができます。また、NSCをから分化したニューロンはまた、繊毛輸送アッセイのために使用することができます。
ニューロスフェアはneuraの増殖から生じる浮遊細胞のクラスターで構成されています特定の増殖因子と非接着面43、44の存在下で増殖L幹/前駆細胞。ニューロスフェアは、正常な発達と病気31、45、46、47の神経幹/前駆細胞を研究するためにin vitro培養モデルのように重要な役割を果たす。ここでは、神経幹/前駆細胞を培養するためにニューロン/グリアへの分化のためのニューロスフェアベースのアッセイを説明します。我々は、特に神経幹/前駆細胞および分化したニューロン（ 図1）の繊毛へのコンポーネントのシグナリングの輸送を強調する。一次ニューロンの培養とは対照的に、一次ニューロスフェアは、培養するのが比較的容易である複数の継代および凍結 – 融解のサイクルに適している、およびニューロン/グリアへの分化を受けることができます。重要なのは、我々はそのニューロスフェア由来神経を決定しました幹/前駆細胞と分化したニューロンは、培養中の繊毛およびこれらの区画における繊毛機能に関連するシグナル伝達分子局在化しています。ニューロスフェアベースの培養方法は、NSCの分化及びニューロンにおいて繊毛形成と繊毛輸送を研究するための理想的なモデル系として役立つことができます。
Protocol
Representative Results
Discussion
ここでは、成体マウスのSVZからニューロスフェア培養を生成し、維持するための方法について説明します。次のように文化に関するいくつかの適切なポイントがあります。 200ミクロン – まず、球の大きさは、通常50の間です。ニューロスフェアは、直径がより大きく300μm以下を取得し、私たちの経験では、継代のための最適な時間が見逃されています。これらの大きな球は、コア内の死細胞?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Work in S.M.’s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).
Materials
| 12 mm round cover glass | Fisherbrand | 12-545-80 | |
| 24 well plate | Falcon | 353047 | |
| 4 % paraformaldehyde | Affymetrix | 19943 | |
| 50 ml tube | Falcon | 352098 | |
| 95 mm X 15 mm petri dish, slippable lid | Fisherbrand | FB0875714G | 10 cm dish |
| 70 µm cell strainer | Falcon | 352350 | |
| Alexa Fluor 488 Affinipure Donkey Anti-Rabbit IgG (H+L) | Jackson Immunoresearch | 711-545-152 | Donkey anti Rabbit, Alexa 488 secondary antibody |
| Arl13B, Clone N295B/66 | Neuromab | AB_11000053 | |
| B-27 Supplement (50X), serum free | ThermoFisher Scientific | 17504001 | B27 |
| Centrifuge | Thermo scientific | ST 40R | |
| Cryogenic vial | Corning | 430488 | |
| DAPI | Sigma | D9542-10MG | |
| Deoxyribonuclease I from bovine pancrease | Sigma | D5025-15KU | Dnase I |
| Dimethyl sulfoxide | Sigma | D8418-100ML | DMSO |
| Disposable Vinyl Specimen Molds | Sakura Tissue-Tek Cryomold | 4565 | 10 mm X 10 mm X 5 mm |
| Dulbecco's Phosphate Buffered Saline 10×, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture | Sigma | D1408-500ML | PBS |
| Dumont #5 Forceps | Fine science tools | 11254-20 | |
| FBS | Sigma | F9026-500ML | |
| Fluoromount-G solution | Southern Biotech | 0100-01 | mounting solution |
| GFAP | DAKO | Z0334 | |
| Goat anti Mouse IgG1 Secondary Antibody, Alexa Fluor 555 conjugate | ThermoFisher Scientific | A-21127 | Goat anti Mouse IgG1, Alexa 555 secondary antibody |
| Goat anti Mouse IgG2a Secondary Antibody, Alexa Fluor 555 conjugate | ThermoFisher Scientific | A-21137 | Goat anti Mouse IgG2a, Alexa 555 secondary antibody |
| Gpr161 | home made | N/A | |
| human bFGF | Sigma | F0291 | FGF |
| hemocytometer | Hausser Scientific | 0.100 mm deep | improved neubauer |
| Isothesia | Henry Schein | NDC 11695-0500-2 | Isofluorane |
| Laminin from Engelbreth-Holm-Swarm Sarcoma basement membrane | Sigma | L2020 | Laminin |
| L-Glutamine (200mM) | Sigma | G7513 | |
| Lipofectamine 3000 Transfection Reagent | ThermoFisher Scientific | L3000 | |
| Mr. Frosty | Nalgene | 5100-0036 | |
| N-2 supplement (100X) | ThermoFisher Scientific | 17502001 | N2 |
| Neurobasal medium | Gibco | 21103-049 | |
| Normal Donkey Serum | Jackson ImmunoResearch | 017-000-121 | |
| OCT compound | Sakura Tissue-Tek | 4583 | OCT |
| Penicillin-Streptomycin | Sigma | P4333-100ML | |
| Poly-L-lysine | Sigma | P4707 | |
| Recombinant human EGF protein, CF | R and D systems | 236-EG-200 | EGF |
| Scissor | Fine science tools | 14060-10 | |
| Superfrost plus microscope slide | Fisher scientific | 12-550-15 | slides |
| Triton X | Bio-Rad | 161-0407 | |
| Trypsin-EDTA solution (10X) | Sigma | T4174-100 | Trypsin |
| COSTAR 6 Well Plate, With Lid Flat Bottom Ultra-Low Attachment Surface Polystyrene, Sterile | Corning | 3471 | ultra-low binding 6 well plate |
| β-tubulin III | Covance | MMS-435P | TUJ1 |
Referências
- Mukhopadhyay, S., Rohatgi, R. G-protein-coupled receptors, Hedgehog signaling and primary cilia. Semin Cell Dev Biol. 33, 63-72 (2014).
- Goetz, S. C., Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet. 11, 331-344 (2010).
- Guemez-Gamboa, A., Coufal, N. G., Gleeson, J. G. Primary cilia in the developing and mature brain. Neuron. 82, 511-521 (2014).
- Louvi, A., Grove, E. A. Cilia in the CNS: the quiet organelle claims center stage. Neuron. 69, 1046-1060 (2011).
- Rohatgi, R., Milenkovic, L., Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science. 317, 372-376 (2007).
- Corbit, K. C., et al. Vertebrate Smoothened functions at the primary cilium. Nature. 437, 1018-1021 (2005).
- Mukhopadhyay, S., et al. The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell. 152, 210-223 (2013).
- Hilgendorf, K. I., Johnson, C. T., Jackson, P. K. The primary cilium as a cellular receiver: organizing ciliary GPCR signaling. Curr Opin Cell Biol. 39, 84-92 (2016).
- Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. C., Mykytyn, K. Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol Biol Cell. 19, 1540-1547 (2008).
- Berbari, N. F., Lewis, J. S., Bishop, G. A., Askwith, C. C., Mykytyn, K. Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc Natl Acad Sci USA. 105, 4242-4246 (2008).
- Loktev, A. V., Jackson, P. K. Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia. Cell Rep. 5, 1316-1329 (2013).
- Marley, A., Choy, R. W., von Zastrow, M. GPR88 reveals a discrete function of primary cilia as selective insulators of GPCR cross-talk. PLoS One. 8, e70857 (2013).
- Marley, A., von Zastrow, M. DISC1 regulates primary cilia that display specific dopamine receptors. PLoS One. 5, e10902 (2010).
- Omori, Y., et al. Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control. PLoS One. 10, e0128422 (2015).
- Koemeter-Cox, A. I., et al. Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons. Proc Natl Acad Sci USA. 111, 10335-10340 (2014).
- Brailov, I., et al. Localization of 5-HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain. Brain Res. 872, 271-275 (2000).
- Hildebrandt, F. .. ,. ,. T. .. ,. &. a. m. p. ;. K. a. t. s. a. n. i. s. ,. N. .., Benzing, T., Katsanis, N. Ciliopathies. N Engl J Med. 364, 1533-1543 (2011).
- Waters, A. M., Beales, P. L. Ciliopathies: an expanding disease spectrum. Pediatr Nephrol. 26, 1039-1056 (2011).
- Badano, J. L., Mitsuma, N., Beales, P. L., Katsanis, N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 7, 125-148 (2006).
- Novarino, G., Akizu, N., Gleeson, J. G. Modeling human disease in humans: the ciliopathies. Cell. 147, 70-79 (2011).
- Brugmann, S. A., Cordero, D. R., Helms, J. A. Craniofacial ciliopathies: A new classification for craniofacial disorders. Am J Med Genet A. 152A (12), 2995-3006 (2010).
- Valente, E. M., Rosti, R. O., Gibbs, E., Gleeson, J. G. Primary cilia in neurodevelopmental disorders. Nat Rev Neurol. 10, 27-36 (2014).
- Davenport, J. R., et al. Disruption of intraflagellar transport in adult mice leads to obesity and slow-onset cystic kidney disease. Curr Biol. 17, 1586-1594 (2007).
- Zaghloul, N. A., Katsanis, N. Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy. J Clin Invest. 119, 428-437 (2009).
- Einstein, E. B., et al. Somatostatin signaling in neuronal cilia is critical for object recognition memory. J Neurosci. 30, 4306-4314 (2010).
- Wang, Z., Phan, T., Storm, D. R. The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia. J Neurosci. 31, 5557-5561 (2011).
- Berbari, N. F., et al. Hippocampal and cortical primary cilia are required for aversive memory in mice. PLoS One. 9, e106576 (2014).
- Han, Y. G., Alvarez-Buylla, A. Role of primary cilia in brain development and cancer. Curr Opin Neurobiol. 20, 58-67 (2010).
- Han, Y. G., et al. Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med. 15, 1062-1065 (2009).
- Lim, D. A., Alvarez-Buylla, A. The Adult Ventricular-Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis. Cold Spring Harbor perspectives in biology. 8, (2016).
- Shimada, I. S., LeComte, M. D., Granger, J. C., Quinlan, N. J., Spees, J. L. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke. J Neurosci. 32, 7926-7940 (2012).
- Mich, J. K., et al. Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain. eLife. 3, e02669 (2014).
- Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell stem cell. 3, 265-278 (2008).
- Pedersen, L. B., Mogensen, J. B., Christensen, S. T. Endocytic Control of Cellular Signaling at the Primary Cilium. Trends Biochem Sci. , (2016).
- Christensen, S. T., Clement, C. A., Satir, P., Pedersen, L. B. Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling. J Pathol. 226, 172-184 (2012).
- Clement, C. A., et al. TGF-beta signaling is associated with endocytosis at the pocket region of the primary cilium. Cell reports. 3, 1806-1814 (2013).
- Taverna, E., Gotz, M., Huttner, W. B. The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annu Rev Cell Dev Biol. 30, 465-502 (2014).
- Joiner, A. M., Green, W. W., McIntyre, J. C. Primary Cilia on Horizontal Basal Cells Regulate Regeneration of the Olfactory Epithelium. J Neurosci. 35, 13761-13772 (2015).
- Inaba, M., Buszczak, M., Yamashita, Y. M. Nanotubes mediate niche-stem-cell signalling in the Drosophila testis. Nature. 523, 329-332 (2015).
- Peng, T., et al. Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration. Nature. 526, 578-582 (2015).
- Rauhauser, A. A., et al. Hedgehog signaling indirectly affects tubular cell survival after obstructive kidney injury. Am J Physiol: Renal Physiol. 309, F770-F778 (2015).
- Chavez, M., et al. Modulation of Ciliary Phosphoinositide Content Regulates Trafficking and Sonic Hedgehog Signaling Output. Dev Cell. 34, 338-350 (2015).
- Lobo, M. V., et al. Cellular characterization of epidermal growth factor-expanded free-floating neurospheres. J. Histochem. Cytochem. 51, 89-103 (2003).
- Reynolds, B. A., Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 255, 1707-1710 (1992).
- Pastrana, E., Silva-Vargas, V., Doetsch, F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell stem cell. 8, 486-498 (2011).
- Nishino, J., Kim, I., Chada, K., Morrison, S. J. Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell. 135, 227-239 (2008).
- Shimada, I. S., Peterson, B. M., Spees, J. L. Isolation of locally derived stem/progenitor cells from the peri-infarct area that do not migrate from the lateral ventricle after cortical stroke. Stroke. 41, e552-e560 (2010).
- Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H., Alvarez-Buylla, A. The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp. , (2010).
- Coles-Takabe, B. L., et al. Don’t look: growing clonal versus nonclonal neural stem cell colonies. Stem Cells. 26, 2938-2944 (2008).
- Pal, K., et al. Smoothened determines beta-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium. J Cell Biol. 212, 861-875 (2016).
- Caspary, T., Larkins, C. E., Anderson, K. V. The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell. 12, 767-778 (2007).
- Bangs, F. K., Schrode, N., Hadjantonakis, A. K., Anderson, K. V. Lineage specificity of primary cilia in the mouse embryo. Nat Cell Biol. 17, 113-122 (2015).
- Berbari, N. F., Bishop, G. A., Askwith, C. C., Lewis, J. S., Mykytyn, K. Hippocampal neurons possess primary cilia in culture. J Neurosci Res. 85, 1095-1100 (2007).
- Paridaen, J. T., Wilsch-Brauninger, M., Huttner, W. B. Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell. 155, 333-344 (2013).
- Kiprilov, E. N., et al. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery. J Cell Biol. 180, 897-904 (2008).
- Mukhopadhyay, S., et al. TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia. Genes Dev. 24, 2180-2193 (2010).
- Ishikawa, H., Marshall, W. F. Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia. Methods Cell Biol. 127, 189-201 (2015).
