Transcriptional Remodeling in Primary Hippocampal Astrocytes from an Alzheimer’s Disease Mouse Model

Tapella, Laura; Gregnanin, Ilaria; Stevano, Alessio; Chiorino, Giovanna; Canonico, P.L.; Distasi, Carla; Genazzani, Armando A.; Lim, Dmitry

doi:10.2174/1567205015666180613113924

Cited by 15 publications

(21 citation statements)

References 43 publications

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“…As shown in Fig. 6a, all 14 genes followed the changes found previously in primary astrocytes 40 . A plot in Fig.…”

Section: Resultssupporting

confidence: 80%

“…This allowed us to avoid the inter-clonal heterogeneity that is a characteristic of clonal selection 48–50 . Validity of this approach was efficiently demonstrated in gene expression and proteomics analyses, in which four independently generated iAstro lines for both WT and 3Tg-AD genotypes were analyzed and gave consistent results, and also demonstrated that 3Tg-iAstro retains the differences as compared with WT-iAstro found previously in primary cultures 40 .…”

Section: Discussionmentioning

confidence: 55%

“…We recently performed a whole-genome microarray analysis of purified primary hippocampal astrocytes 40 in which bioinformatic analysis revealed expression differences between astrocytes prepared from WT and from 3Tg animals. To further characterize our immortalized cell lines we therefore primed 14 of the identified genes that showed modifications (Car9, P2ry2, Padi2, Col2a1, Col4a6, Fbln2, Cacna2d3, Panx1, Pcdh17, Trim12, Ccl27a PESKY, Ptchd2, Slc2a4 and Samd4).…”

Section: Resultsmentioning

confidence: 99%

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Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model

Rocchio¹,

Tapella²,

Manfredi³

et al. 2019

Cell Death Dis

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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 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.

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“…As shown in Fig. 6a, all 14 genes followed the changes found previously in primary astrocytes 40 . A plot in Fig.…”

Section: Resultssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model

Rocchio¹,

Tapella²,

Manfredi³

et al. 2019

Cell Death Dis

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show abstract

“…Recently, we have generated and characterized immortalized astrocytic cell lines from hippocampi of 3xTg-AD mice, a popular and well-studied AD mouse model 27 . These lines, named WT-and 3xTg-AD immortalized astrocytes (WT-iAstro and 3Tg-iAstro) recapitulate the features of primary astrocytic cultures from 3xTg-AD mice in terms of gene profiling, protein expression and Ca 2+ signaling 27,28 . Here we used WT-iAstro and 3Tg-iAstro lines to study mitochondrial functions and their association with Ca 2+ signaling, mito-ER interaction and proteostasis.…”

Section: Introductionmentioning

confidence: 98%

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice

et al. 2020

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The pathogenesis of Alzheimer's disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca 2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER-and mitochondria-targeted aequorin-based Ca 2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca 2+ while Ca 2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8-10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca 2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

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“…Virtually no microglial cells have been detected in purified astroglial cultures after MACS procedure as was assessed by immunostaining with anti-Iba1 antibody (1:500, D.B.A., Segrate, Italy, Cat. 019-19741) [ 81 ].…”

Section: Methodsmentioning

confidence: 99%

Neuronal Activity-Dependent Activation of Astroglial Calcineurin in Mouse Primary Hippocampal Cultures

Lim

Mapelli

Canonico

et al. 2018

IJMS

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Astrocytes respond to neuronal activity by generating calcium signals which are implicated in the regulation of astroglial housekeeping functions and/or in modulation of synaptic transmission. We hypothesized that activity-induced calcium signals in astrocytes may activate calcineurin (CaN), a calcium/calmodulin-regulated protein phosphatase, implicated in neuropathology, but whose role in astroglial physiology remains unclear. We used a lentiviral vector expressing NFAT-EYFP (NY) fluorescent calcineurin sensor and a chemical protocol of LTP induction (cLTP) to show that, in mixed neuron-astrocytic hippocampal cultures, cLTP induced robust NY translocation into astrocyte nuclei and, hence, CaN activation. NY translocation was abolished by the CaN inhibitor FK506, and was not observed in pure astroglial cultures. Using Fura-2 single cell calcium imaging, we found sustained Ca2+ elevations in juxtaneuronal, but not distal, astrocytes. Pharmacological analysis revealed that both the Ca2+ signals and the nuclear NY translocation in astrocytes required NMDA and mGluR5 receptors and depended on extracellular Ca2+ entry via a store-operated mechanism. Our results provide a proof of principle that calcineurin in astrocytes may be activated in response to neuronal activity, thereby delineating a framework for investigating the role of astroglial CaN in the physiology of central nervous system.

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Transcriptional Remodeling in Primary Hippocampal Astrocytes from an Alzheimer’s Disease Mouse Model

Abstract: Our data show that in cultured astrocytes from an AD mouse model, transcriptional changes occur and are different from those reported in models mimicking later stages of the disease.

Cited by 15 publications

References 43 publications

Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model

Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice

Neuronal Activity-Dependent Activation of Astroglial Calcineurin in Mouse Primary Hippocampal Cultures

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