Grove J, Gomez J, Kentroti S, Vernadakis A
Department of Psychiatry, University of Colorado Health Sciences Center, Denver 80206, USA.
Brain Res Bull. 1996;39(4):211-7. doi: 10.1016/0361-9230(95)02130-2.
This study was targeted at the beginning to understand the functional status of glial cells derived from aged brain. We have previously characterized passaged cell cultures derived from aged mouse cerebral hemispheres (MACH) and found them to contain large populations of astrocytes, type 1, as well as limited numbers of astrocytes, type 2, oligodendrocytes, and progenitor cells. Using the activity of the astrocyte marker, glutamine synthetase (GS), as an index, we found that MACH astrocytes continue to respond to several microenvironmental signals, including the cAMP-enhancing agents dibutyryl cAMP and R020-1724 (an inhibitor of phosphodiesterase). In addition, whereas the basal activity of GS increased with cell passage, their response to these agents was cell-passage dependent, increasing at early (21-22) passages and decreasing at later (46-51) passages. Because neurotrophins (i.e., NGF and EGF) also provide microenvironmental signals essential to normal glial function, MACH cultures were assessed for their response to these factors. MACH cultures at passage 35 responded to treatment with NGF and EGF with a dose-dependent increase in GS activity by both neurotrophins. With the intention of arresting these cultures at a specific stage of differentiation, these cells were immortalized at passage 19 by transfection with the gene encoding SV40 Large T antigen. These immortalized MACH responded to exposure to dBcAMP and RO20-1724 with a marked decrease in GS activity, mimicking the response of normal MACH glia at late passage. Finally, because it has been shown that glia from both immature and adult brain contain neurotrophins and respond to neurotrophins via a receptor-mediated pathway, we examined expression of NGF protein as well as NGF (p75) and EGF receptor protein in various passages and colonies of normal and immortalized MACH cultures. We found a consistent expression of all three proteins in the various cell populations. Results of this study suggest that astrocytes from aging brain continue to function normally with respect to several parameters (i.e., response to neurotrophins and differentiating agents). Thus, they retain their plasticity to a great degree through early cell passages. However, with advancing cell passage this plasticity declines and cell homeostasis is impaired. We propose, therefore, that astrocytes undergo several critical periods in their functional lifespan, one of which is represented by the functional transition demonstrated in this study.
本研究从一开始就旨在了解源自老年大脑的神经胶质细胞的功能状态。我们之前对源自老年小鼠大脑半球（MACH）的传代细胞培养物进行了表征，发现它们含有大量的1型星形胶质细胞，以及数量有限的2型星形胶质细胞、少突胶质细胞和祖细胞。以星形胶质细胞标志物谷氨酰胺合成酶（GS）的活性为指标，我们发现MACH星形胶质细胞继续对几种微环境信号作出反应，包括环磷酸腺苷增强剂二丁酰环磷酸腺苷和R020 - 1724（一种磷酸二酯酶抑制剂）。此外，虽然GS的基础活性随着细胞传代而增加，但其对这些试剂的反应是细胞传代依赖性的，在早期传代（21 - 22代）时增加，而在后期传代（46 - 51代）时降低。因为神经营养因子（即神经生长因子和表皮生长因子）也提供对正常神经胶质细胞功能至关重要的微环境信号，所以对MACH培养物对这些因子的反应进行了评估。第35代的MACH培养物对神经生长因子和表皮生长因子的处理有反应，两种神经营养因子均使GS活性呈剂量依赖性增加。为了使这些培养物在特定的分化阶段停滞，在第19代时通过用编码SV40大T抗原的基因转染使这些细胞永生化。这些永生化的MACH对暴露于二丁酰环磷酸腺苷和RO20 - 1724的反应是GS活性显著降低，这与晚期传代的正常MACH神经胶质细胞的反应相似。最后，因为已经表明来自未成熟和成年大脑的神经胶质细胞都含有神经营养因子并通过受体介导的途径对神经营养因子作出反应，所以我们检测了正常和永生化MACH培养物的不同传代和集落中神经生长因子蛋白以及神经生长因子（p75）和表皮生长因子受体蛋白的表达。我们发现在各种细胞群体中这三种蛋白都有一致的表达。本研究结果表明，来自衰老大脑的星形胶质细胞在几个参数方面（即对神经营养因子和分化剂的反应）继续正常发挥功能。因此，它们在早期细胞传代过程中在很大程度上保留了其可塑性。然而，随着细胞传代的推进，这种可塑性下降，细胞内稳态受到损害。因此，我们提出星形胶质细胞在其功能寿命中经历几个关键时期，其中之一由本研究中证明的功能转变所代表。
