Dataset Information

Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP-activated protein kinase and induces breakdown of glycogen

ABSTRACT: Astrocytes contain the majority of the brain’s supply of glycogen which has been suggested to play an important role in neuronal synaptic plasticity. This may be mediated by release of glycogen-derived lactate or glutamine supporting neuronal metabolism or via induction of neuronal signaling pathways. The second messenger cyclic AMP (cAMP) can induce glycogen breakdown and activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to be the link between neuronal depolarization and glycogen breakdown. However, recent studies have revealed that sAC residing in the mitochondria rather than the cytosol is regulating mitochondrial bioenergetics, and that employing pharmacological inhibitors of sAC significantly alters cellular energy metabolism. This effect on energy metabolism could influence the interpretation of previous studies employing pharmacological inhibition of sAC. Here, we show that pharmacological inhibition of sAC lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMPK, and decreases glycogen stores in cultured astrocytes. In light of this, we discuss the pharmacological challenges of investigating the functional and metabolic roles of sAC in astrocytes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Acyrthosiphon Pisum (pea Aphid) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Astrocyte

SUBMITTER: Martin R. Larsen

LAB HEAD: Martin R. Larsen

PROVIDER: PXD027901 | Pride | 2021-08-13

REPOSITORIES: pride

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Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP-activated protein kinase and induces breakdown of glycogen.

Jakobsen Emil E Andersen Jens V JV Christensen Sofie K SK Siamka Olga O Larsen Martin R MR Waagepetersen Helle S HS Aldana Blanca I BI Bak Lasse K LK

Glia 20210811 12

Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulato ...[more]

PMID: 34378239

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Project description:Brain inflammation, a common feature in neurodegenerative diseases, is a complex series of events, which can be detrimental and even lead to neuronal death. Nonetheless, several studies suggest that inflammatory signals are also positively influencing neural cell proliferation, survival, migration and differentiation. Recently, correlative studies suggested that astrocytes are able to dedifferentiate upon injury, and may thereby re-acquire neural stem cells (NSC) potential. However, the mechanism underlying this dedifferentiation process upon injury remains unclear. In this study, we find that during the early response of reactive gliosis, inflammation induces a conversion of mature astrocytes into neural progenitors. A TNF treatment induces the decrease of specific astrocyte markers, such as GFAP or genes related to glycogen metabolism, while a subset of these cells re-express immaturity markers, such as CD44, Musashi-1 and Oct4. Thus, TNF treatment results in the appearance of cells that exhibit a neural progenitor phenotype and are able to proliferate and differentiate into neurons and/or astrocytes. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway. Cultures of primary mouse astrocytes were treated with TNF (50 ng/mL) for 24 hours. Cells were collected and immediately homogenized in cooled down RNA NOW reagent (OZYME). Total RNA was extracted according to RNA NOW manufacturer's recommendations with -20°C overnight incubation for small RNA precipitation. Total RNA integrity and purity were assessed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kits (Agilent Technologies). Only good-quality RNA (no contamination or degradation, RIN > 9) was used and further processed. Total RNA samples were reverse-transcribed to double-stranded cDNA using specific primers, which reduce the priming of rRNA. cRNA was generated by in vitro transcription and reverse transcribed into a sense single-stranded cDNA. The cDNA was fragmented, labeled, and hybridized onto Affymetrix GeneChip Mouse Gene 1.0 ST Arrays according to the Ambion Whole Transcript Expression kit for Affymetrix GeneChip Whole Transcript Expression Array Protocol (P/N 4425209 Rev.B 05/2009) and GeneChip WT Terminal Labeling and Hybridization User Manual for use with the Ambion Whole Transcript Expression kit (P/N 702808 Rev.6). Microarrays were then washed, stained, and scanned according to the manufacturer's instructions. The samples cover combinations of 7 time points (0H, 6H, 24H, 48H, 72H, 1W, 2W) and two conditions (TNF and control), with multiple replicates per condition and time point.

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