Immortalization and Characterization of GFAP-expressing Glial Cells from the Adult Mouse Hypothalamus, Cortex, and Brain Stem

Abstract

The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1, Aqp4, Glt1 and S100b, and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b, Tnfα and Cxcl5 in mHypoA-Ast1 cells after 4 h, while 50 μM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research.