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Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response.

ABSTRACT: Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ? 2 and p ? 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response.

SUBMITTER: Allen SP

PROVIDER: S-EPMC7672558 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response.

Allen Scott P SP Seehra Rajpinder Singh RS Heath Paul R PR Hall Benjamin P C BPC Bates Jessica J Garwood Claire J CJ Matuszyk Martyna M MM Wharton Stephen B SB Simpson Julie E JE

International journal of molecular sciences 20201028 21

Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under nor ...[more]

PMID: 33126586

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Project description:Epidemiological and clinical studies have long shown that exposure to high levels of heavy metals are associated with increased risks of neurodegenerative diseases. It is widely accepted that autophagic dysfunction is involved in pathogenesis of various neurodegenerative disorders; however, the role of heavy metals in regulation of macroautophagy/autophagy is unclear. Here, we show that manganese (Mn) induces a decline in nuclear localization of TFEB (transcription factor EB), a master regulator of the autophagy-lysosome pathway, leading to autophagic dysfunction in astrocytes of mouse striatum. We further show that Mn exposure suppresses autophagic-lysosomal degradation of mitochondria and induces accumulation of unhealthy mitochondria. Activation of autophagy by rapamycin or TFEB overexpression ameliorates Mn-induced mitochondrial respiratory dysfunction and reactive oxygen species (ROS) generation in astrocytes, suggesting a causal relation between autophagic failure and mitochondrial dysfunction in Mn toxicity. Taken together, our data demonstrate that Mn inhibits TFEB activity, leading to impaired autophagy that is causally related to mitochondrial dysfunction in astrocytes. These findings reveal a previously unappreciated role for Mn in dysregulation of autophagy and identify TFEB as a potential therapeutic target to mitigate Mn toxicity.AbbreviationsBECN1: beclin 1; CTSD: cathepsin D; DMEM: Dulbecco's Modified Eagle Medium; GFAP: glial fibrillary acid protein; GFP: green fluorescent protein; HBSS: hanks balanced salt solution; LAMP: lysosomal-associated membrane protein; LDH: lactate dehydrogenase; Lys Inh: lysosomal inhibitors; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; Mn: manganese; MTOR: mechanistic target of rapamycin kinase; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PI: propidium iodide; ROS: reactive oxygen species; s.c.: subcutaneous; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB.

Project description:BACKGROUND:Parkinson's disease (PD) is one of the neurodegeneration diseases characterized by the gradual loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. Substantial evidence indicates that at the cellular level mitochondrial dysfunction is a key factor leading to pathological features such as neuronal death and accumulation of misfolded ?-synuclein aggregations. Autologous transplantation of healthy purified mitochondria has shown to attenuate phenotypes in vitro and in vivo models of PD. However, there are significant technical difficulties in obtaining large amounts of purified mitochondria with normal function. In addition, the half-life of mitochondria varies between days to a few weeks. Thus, identifying a continuous source of healthy mitochondria via intercellular mitochondrial transfer is an attractive option for therapeutic purposes. In this study, we asked whether iPSCs derived astrocytes can serve as a donor to provide functional mitochondria and rescue injured DA neurons after rotenone exposure in an in vitro model of PD. METHODS:We generated DA neurons and astrocytes from human iPSCs and hESCs. We established an astroglial-neuronal co-culture system to investigate the intercellular mitochondrial transfer, as well as the neuroprotective effect of mitochondrial transfer. We employed immunocytochemistry and FACS analysis to track mitochondria. RESULTS:We showed evidence that iPSCs-derived astrocytes or astrocytic conditioned media (ACM) can rescue DA neurons degeneration via intercellular mitochondrial transfer in a rotenone induced in vitro PD model. Specifically, we showed that iPSCs-derived astrocytes from health spontaneously release functional mitochondria into the media. Mito-Tracker Green tagged astrocytic mitochondria were detected in the ACM and were shown to be internalized by the injured neurons via a phospho-p38 depended pathway. Transferred mitochondria were able to significantly reverse DA neurodegeneration and axonal pruning following exposure to rotenone. When rotenone injured neurons were cultured in presence of ACM depleted of mitochondria (by ultrafiltration), the neuroprotective effects were abolished. CONCLUSIONS:Our studies provide the proof of principle that iPSCs-derived astrocytes can act as mitochondria donor to the injured DA neurons and attenuate pathology. Using iPSCs derived astrocytes as a donor can provide a novel strategy that can be further developed for cellular therapy for PD.

Dataset Information

Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response.

Publications

Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response.

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