Extracellular Matrix Proteins Differentiate Postnatal Mouse Retina Neurospheres into Neurons or Glia Profiles

IF 3.8 3区 医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Orquidia Guadalupe Méndez-Flores, Anna Carolina Rego Costa, Andrey Fabiano Lourenço de Aguiar, Yolanda Paes-Colli, Cláudia Maria Batista, Victor T. Ribeiro-Resende, Arturo Ortega, Ricardo A. de Melo Reis
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引用次数: 0

Abstract

The mammalian retinal progenitor cells (RPC) exit the cell cycle through signaling of intrinsic and extrinsic factors and give rise to several types of neurons and Müller glia, following an organized spatial–temporal pattern. Extracellular matrix (ECM) plays an important role in retinal development, influencing RPC proliferation and differentiation into pro-gliogenic and/or neurogenic phenotypes. Here, we investigated how four different ECM constituents, fibronectin, vitronectin, collagen type IV and laminin-1 (α1β1γ1), added on coverslips previously treated with 10 µg/mL poly-L-lysine, could impact differentiation of retinal neurospheres generated with epidermal growth factor (EGF) 20 ng/mL and cultivated for four days. Progenitors (activated by muscimol, a GABAA agonist), neurons (by KCl and/or AMPA, a glutamatergic agonist) and Müller glia (by ATP) show distinct functional responses in terms of calcium imaging due to the pattern of selective receptors and channels expressed during development. A highly heterogeneous cell population was generated when neurospheres were cultivated in different ECM molecules, suggesting the presence of high, medium, and low-responsive cells. As shown, collagen type IV or laminin-1 for 6 days in DMEM F12 had similar responses, revealing that nearly 55% of cells were responsive to KCl, 28–39% to AMPA, 18–28% to ATP and almost none to muscimol (less than 0.5%). On the other hand, in the presence of fibronectin, 56% of retinal neurospheres were induced to respond to KCl, 32% to AMPA, 33% to ATP and 2.8% to muscimol. Finally, neurospheres raised in vitronectin had around 67% of cells responsive to KCl, 41% to AMPA, less than 20% to ATP and 3% to muscimol. As expected, differentiated cells in the presence of fibronectin were immuno-labelled and expressed higher levels of glial fibrillary acidic protein (GFAP), compared to other substrates, while cultures prepared in the presence of vitronectin had increased expression of neuron-specific class III β-tubulin (TUJ-1), a neuronal marker. Altogether, our data suggest that, compared to laminin, a standard substrate, collagen and vitronectin increased the number of functional neurons, while fibronectin induced a two-fold increase in the number of glial cells in the developing cells of the mice retina.

Abstract Image

细胞外基质蛋白将小鼠视网膜神经球分化为神经元或神经胶质。
哺乳动物视网膜祖细胞(RPC)通过内在和外在因素的信号传导退出细胞周期,并根据有组织的时空模式产生几种类型的神经元和神经胶质。细胞外基质(ECM)在视网膜发育中起重要作用,影响RPC的增殖和向前胶质细胞和/或神经细胞表型的分化。在此,我们研究了四种不同的ECM成分,纤维连接蛋白,玻璃体连接蛋白,IV型胶原蛋白和层粘连蛋白-1 (α1β1γ1),添加到先前用10µg/mL聚l -赖氨酸处理的盖片上,如何影响表皮生长因子(EGF) 20 ng/mL培养4天产生的视网膜神经球的分化。祖细胞(由GABAA激动剂muscimol激活)、神经元(由KCl和/或谷氨酸受体激动剂AMPA激活)和突触神经胶质(由ATP激活)在钙成像方面表现出不同的功能反应,这是由于在发育过程中表达的选择性受体和通道的模式。当神经球在不同的ECM分子中培养时,产生了高度异质的细胞群,表明存在高、中、低反应细胞。如图所示,IV型胶原或laminin-1在DMEM F12中作用6天后有类似的反应,表明近55%的细胞对KCl有反应,对AMPA有28-39%的反应,对ATP有18-28%的反应,对muscimol几乎没有反应(小于0.5%)。另一方面,在纤维连接蛋白存在的情况下,56%的视网膜神经球对KCl、32%对AMPA、33%对ATP和2.8%对muscimol有反应。最后,在玻璃质粘连蛋白中培养的神经球中,约67%的细胞对KCl有反应,41%对AMPA有反应,不到20%对ATP有反应,3%对muscimol有反应。正如预期的那样,与其他底物相比,在纤维连接蛋白存在下的分化细胞被免疫标记并表达更高水平的胶质纤维酸性蛋白(GFAP),而在玻璃体连接蛋白存在下制备的培养物增加了神经元特异性III类β-微管蛋白(TUJ-1)的表达,这是一种神经元标志物。总的来说,我们的数据表明,与层粘连蛋白(一种标准底物)相比,胶原蛋白和玻璃体连接蛋白增加了功能神经元的数量,而纤维连接蛋白诱导小鼠视网膜发育细胞中胶质细胞的数量增加了两倍。
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来源期刊
Neurochemical Research
Neurochemical Research 医学-神经科学
CiteScore
7.70
自引率
2.30%
发文量
320
审稿时长
6 months
期刊介绍: Neurochemical Research is devoted to the rapid publication of studies that use neurochemical methodology in research on nervous system structure and function. The journal publishes original reports of experimental and clinical research results, perceptive reviews of significant problem areas in the neurosciences, brief comments of a methodological or interpretive nature, and research summaries conducted by leading scientists whose works are not readily available in English.
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