Sun Wei, Cornwell Adam, Li Jiashu, Peng Sisi, Osorio M Joana, Aalling Nadia, Wang Su, Benraiss Abdellatif, Lou Nanhong, Goldman Steven A, Nedergaard Maiken
Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, New York 14642, and
Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, New York 14642, and.
J Neurosci. 2017 Apr 26;37(17):4493-4507. doi: 10.1523/JNEUROSCI.3199-16.2017. Epub 2017 Mar 23.
Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as such by their expression of glial fibrillary acidic protein, glutamine synthetase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathognomonic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9+ cells with GLT1+ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple mini-strokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immunolabeling. The analysis showed that SOX9+ astrocytes constitute ∼10-20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain. Astrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain.
近年来，星形胶质细胞已成为密集实验研究的焦点，然而用于其明确鉴定的标志物仍然稀少且不完善。星形胶质细胞可通过其胶质纤维酸性蛋白、谷氨酰胺合成酶、谷氨酸转运体1（GLT1）、水通道蛋白-4、醛脱氢酶1家族成员L1及其他蛋白质的表达而被识别。然而，这些蛋白质在发育和功能上均可受到调控，限制了它们的实用性。为了鉴定星形胶质细胞表型的特征性核标志物，我们评估了经荧光激活细胞分选（FACS）纯化的成年星形胶质细胞的差异RNA表达，并在此基础上评估了转录因子SOX9在小鼠和人类大脑中的表达。我们发现，在成人大脑中，除室管膜细胞外，SOX9几乎仅由星形胶质细胞表达；而在神经发生区域，神经祖细胞也表达SOX9。对SOX9+细胞与GLT1+细胞的转录组比较显示，这两类细胞群体表现出很大程度的基因表达重叠。SOX9的表达在衰老过程中并未降低，反而在多种情况下被反应性星形胶质细胞上调，包括肌萎缩侧索硬化症（SOD1G93A）小鼠模型、大脑中动脉闭塞和多次小中风。我们使用各向同性分选技术结合SOX9免疫标记对星形胶质细胞的相对数量进行了定量。分析表明，在大多数中枢神经系统区域，SOX9+星形胶质细胞约占细胞总数的10%-20%，占细胞总数的比例比正常成人大脑中先前估计的要小。传统上，星形胶质细胞是通过针对细胞质或质膜中细胞特异性抗原的抗体进行免疫组织化学鉴定的。我们在此表明，在神经发生区域以外的中枢神经系统所有主要区域，SOX9是星形胶质细胞特异性的核标志物。基于SOX9免疫标记，我们证明，在正常成年小鼠大脑中，星形胶质细胞占细胞总数的比例比先前估计的要小。
