Rocchio et al. Cell Death and Disease (2019) 10:24 https://doi.org/10.1038/s41419-018-1264-8 Cell Death & Disease ARTICLE Open Access Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model Francesca Rocchio1,5, Laura Tapella1, Marcello Manfredi2,3, Mariangela Chisari4, Francesca Ronco1, Federico Alessandro Ruffinatti1, Eleonora Conte2, Pier Luigi Canonico1, Maria Angela Sortino4, Mariagrazia Grilli 1, Emilio Marengo2, Armando A. Genazzani1 and Dmitry Lim 1 Abstract Evidence is rapidly growing regarding a role of astroglial cells in the pathogenesis of Alzheimer’s disease (AD), and the hippocampus is one of the important brain regions affected in AD. While primary astroglial cultures, both from wild- type mice and from rodent models of AD, have been useful for studying astrocyte-specific alterations, the limited cell number and short primary culture lifetime have limited the use of primary hippocampal astrocytes. To overcome these limitations, we have now established immortalized astroglial cell lines from the hippocampus of 3xTg-AD and wild- type control mice (3Tg-iAstro and WT-iAstro, respectively). Both 3Tg-iAstro and WT-iAstro maintain an astroglial phenotype and markers (glutamine synthetase, aldehyde dehydrogenase 1 family member L1 and aquaporin-4) but display proliferative potential until at least passage 25. Furthermore, these cell lines maintain the potassium inward 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; rectifying (Kir) current and present transcriptional and proteomic profiles compatible with primary astrocytes. Importantly, differences between the 3Tg-iAstro and WT-iAstro cell lines in terms of calcium signaling and in terms of transcriptional changes can be re-conducted to the changes previously reported in primary astroglial cells. To illustrate the versatility of this model we performed shotgun mass spectrometry proteomic analysis and found that proteins related to RNA binding and ribosome are differentially expressed in 3Tg-iAstro vs WT-iAstro. In summary, we present here immortalized hippocampal astrocytes from WT and 3xTg-AD mice that might be a useful model to speed up research on the role of astrocytes in AD. Introduction neuroinflammation, a growing body of evidence suggests While in Alzheimer’s disease (AD) astrocytes have been that astroglial alterations occur in the early stages of AD, historically associated with reactive gliosis and compromising their housekeeping and homeostatic functions that in turn may result in synaptic and neuronal malfunction1,2. Correspondence: Armando A. Genazzani ([email protected])or Most of the information about the role of astrocytes in Dmitry Lim ([email protected]) brain pathology has been collected in in vitro experiments 1Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale, Novara, Italy on primary cultures. Easy to prepare and handle, astroglial 2Department of Sciences and Technological Innovation, Università degli Studi primary cultures are made from different brain areas3,4 – del Piemonte Orientale, Alessandria, Italy and different animal species5 8. However, primary cul- Full list of author information is available at the end of the article. These authors contributed equally: Francesca Rocchio, Laura Tapella tures present several limitations such as inter-culture Edited by A. Verkhratsky © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Rocchio et al. Cell Death and Disease (2019) 10:24 Page 2 of 18 variability, short culture lifetime and limited number of were confirmed for morphological identity to other lines, cells if cultures are prepared from specific brain areas, but were maintained as backups and have not been such as the hippocampus. To overcome these limitations, characterized (#1 and #5 for WT-iAstro and #1 and #4 for immortalized astroglial lines have been proposed. The 3Tg-iAstro lines). first attempts to generate a permanent astroglial cell line, iAstro cells show a morphology similar and virtually not deriving from brain tumors, date back to early eigh- indistinguishable to those of primary hippocampal astro- ties9. Since then, a number of immortalized astroglial lines cytes in bright field microscopy (Fig. 1a). We also eval- – have been established10 23. Most of them derive from a uated the expression of the astroglial markers aquaporin-4 highly heterogeneous population of cortical primary (AQP4), glutamine synthetase (GS) and aldehyde dehy- astrocytes; however, immortalization of astrocytes from drogenase 1 family member l1 (Aldh1l1) (Fig. 1b) by brain regions other than cortex have also been reported, immunocytochemistry. Importantly, we found expression e.g., from the cerebellum9 or from the midbrain21. Sur- of all three markers in all cell lines. Immunocytochemical prisingly, few attempts have been reported to immortalize analysis for glial fibrillary acidic protein (GFAP) showed, astrocytes from hippocampus and also from animal instead, that only a small proportion of cells were positive models of AD. In this regard, Morikawa et al.18 have in the established immortalized cell lines (15.4 ± 5.3 % in generated immortalized astrocytes from ApoE2, ApoE3 WT-iAstro vs 16.7 ± 5.9 % in 3Tg-iAstro cells, p > 0.05) and ApoE4 knock-in mice. (Fig. 1c), while 100% of cells were GFAP positive in WT- In this report we introduce immortalized astroglial lines and 3Tg-primary astrocytes. from the hippocampus of a well-characterized AD mouse We also decided to have a quantitative measure of the model, 3xTg-AD, and from wild-type (WT) control mice, expression of the three markers by evaluating protein from now on referred to as 3Tg-iAstro and WT-iAstro, levels in western blotting on three of the four cell lines. respectively. WT-iAstro cell lines show features of pri- The three markers were mostly decreased compared to mary hippocampal astrocytes such as basic electro- primary cell lines, albeit at different levels. AQP4 was the physiological properties, and a similar transcriptional and least decreased, while Aldh1l1 was the most decreased, proteomic profile. More importantly, 3Tg-iAstro show with qualitative consistency in the immortalized cell lines + alterations in transcription and deregulation of Ca2 sig- tested. Importantly, not only were all the markers naling as it was reported for its primary counterparts. To detectable, but they were also represented at similar levels illustrate the versatility of this model we also performed between the WT- and 3Tg-iAstro cell lines (Fig. 2). shotgun mass spectrometry proteomic analysis and found We also evaluated the ability of the iAstro cell lines to that proteins related to RNA binding and ribosome are be passaged in culture. iAstro lines did not change their differentially expressed in 3Tg-iAstro vs WT-iAstro. morphology or marker expression significantly at least up These data demonstrate that iAstro lines represent a to 20th passage (not shown). versatile and useful cellular model to investigate astroglial AD-related pathobiology. Kir currents are present but are not different in WT-iAstro and 3Tg-iAstro Results Maintenance of ionic balance is one of the house- Generation of immortalized hippocampal astrocytes keeping functions of astrocytes and a feature which might (iAstro) from WT and 3xTg-AD mice have been lost during immortalization. Potassium buf- + Six immortalized cell lines from WT (WT-iAstro#1–6) fering by inwardly rectifying K (Kir) channels during and from 3xTg-AD (3Tg-iAstro#1–6) mice were gener- neuronal activity is fundamental to maintain adequate ated, from separate primary astrocyte cell cultures. For synaptic transmission and neuronal excitability24.We immortalization, primary astroglial cultures were first have previously shown that Kir channels are functionally depleted of microglial cells by magnetic-assisted cell expressed in primary hippocampal astrocytes25. There- sorting (MACS) using anti-CD11b-conjugated microbe- fore, we performed patch-clamp experiments in WT- ads in order to obtain a population of highly purified iAstro#2 and 3Tg-iAstro#2 lines. To confirm that Kir astrocytes. Astrocytes were then transduced using retro- function was not affected by the immortalization process, virus expressing SV40 large T antigen. Transformed cells control hippocampal mouse astrocytes were also patched were selected in G418, amplified and stabilized for 12 in order to record Kir current. Cells were exposed to a passages prior to characterization. No clonal selection was step protocol of increasing voltage (20 mV increments) performed to maintain the natural hererogeneity of the from −180