miR-29a differentially regulates cell survival in astrocytes from cornu ammonis 1 and dentate gyrus by targeting VDAC1
Introduction
Global cerebral ischemia leads to post-resuscitation neurological impairment in survivors. Pyramidal neurons in the cornu ammonis 1 (CA1) region of the hippocampus are selectively sensitive to ischemia, dying in the days following reperfusion. However neurons in the adjacent dentate gyrus (DG) have a relatively higher ischemic resistance and survive (for review, Ouyang et al., 2014). Delayed neuronal death in the CA1 occurs secondary to disruption in mitochondrial function (Owens et al., 2015), inducing release of cytochrome c and other pro-apoptotic factors into the cytoplasm (Ouyang et al., 1999, Niizuma et al., 2009). Delineating the mechanisms that determine observed differences between the CA1 and DG hippocampal subregions in the cellular response to injury might provide new avenues in the development of clinical therapies for ischemic brain injury.
Lack of consideration for other cell types in the brain has been a proposed factor in the translational failure of potential neuroprotective strategies (Nedergaard and Dirnagl, 2005). Astrocytes, the most abundant cell type in the brain, play many key roles supporting normal neuronal functioning, including preserving ionic and acid-base balance, modulating neurotransmission, and maintaining neuronal energy stores (Clarke and Barres, 2013, Barreto et al., 2011). Critically, astrocyte homeostasis is tightly coupled to neuronal cell fate following ischemic injury (Nedergaard and Dirnagl, 2005, Barreto et al., 2011, Ouyang et al., 2014). We have previously observed that both neurons and astrocytes isolated from different brain regions show differential sensitivity to injuries (Xu et al., 2001). We further observed that within the hippocampus CA1 astrocytes were more sensitive to ischemic injury, with greater mitochondrial dysfunction compared to DG astrocytes (Ouyang et al., 2007). Moreover we demonstrated that disruption of mitochondrial homeostasis in resident astrocytes contributes to neuronal cell death in CA1 following transient forebrain ischemia (Xu et al., 2010, Ouyang et al., 2013). However, the factors that determine regional hippocampal differences in post-ischemic astrocyte dysfunction, and therefore neuronal cell fate, remain incompletely understood.
Cell function and fate following stress are determined in part by the interface between gene transcription and epigenetic modifiers of gene expression (Mehler, 2008). MicroRNAs (miRs) are a class of endogenously expressed, non-coding RNAs, which modify gene expression by binding the 3′ untranslated region (UTR) of target genes and inhibiting translation. Numerous miRs are expressed in a cell-specific manner, and miR-29 is selectively enriched in astrocytes (Smirnova et al., 2005). Expression of miR-29a is suppressed in neurodegenerative disorders, including Alzheimer's disease and Huntington's disease (Roshan et al., 2009), and brain-targeted knockdown of miR-29a in developing animals results in neurological dysfunction, notably region-specific hippocampal neuronal cell death (Roshan et al., 2014). We previously observed in an in vivo rodent model of transient global cerebral ischemia that miR-29a increased in the DG, but decreased in the CA1, and that overexpression of miR-29a resulted in protection of CA1 neurons from delayed neuronal death (Ouyang et al., 2013). Further, we observed in cortical astrocyte cultures that increasing levels of miR-29a protected cells from ischemia-like stress, while decreasing levels of miR-29a disrupted mitochondrial homeostasis, resulting in cell death (Ouyang et al., 2013). However, the mechanisms for this effect remain unclear. To further delineate mechanisms of hippocampal regional heterogeneity, which may explain subregion-specific vulnerability, we utilized astrocytes selectively cultured from hippocampal CA1 and DG subregions to investigate the roles of miR-29a, and a mitochondrial target, the voltage-dependent anion channel-1 (VDAC1, Bargaje et al., 2012), in astrocyte cell death following ischemia-like stress.
Section snippets
Cell cultures and transfection
All experimental protocols using animals were performed according to protocols approved by the Stanford University Animal Care and Use Committee and in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Primary hippocampal astrocyte cultures were prepared from postnatal (days 3–4) Swiss Webster mice (Simonsen, Gilroy, CA) as previously described (Xu et al., 2001). Briefly, the left and right hippocampi were identified morphologically and by
Results
In order to verify successful separation of CA1 and DG hippocampal subregions we analyzed astrocyte cultures for relative expression of desmoplakin mRNA, which is selectively expressed in the DG (Lein et al., 2004). Levels of desmoplakin mRNA were 4.0 ± 0.2 fold higher in astrocytes cultured from the DG versus from CA1. No difference was observed in baseline levels of miR-29a expression between CA1 and DG astrocytes (DG = 1.19 ± 0.78 fold versus CA1). Subjecting cultures to GD injury for 48 h resulted
Discussion
The results of the present study are the first to demonstrate a differential response in miR-29a to stress between astrocytes cultured from the more ischemia-sensitive CA1 hippocampal subregion and astrocytes from the DG. We have previously observed that increasing levels of miR-29a in cortical astrocytes preserved mitochondrial homeostasis following ischemia-like stress (Ouyang et al., 2013). In the present study, overexpression of miR-29a in both CA1 and DG subregion astrocytes augmented
Conclusions
Astrocytes cultured from the hippocampal CA1 subregion exhibited a greater decrease in miR-29a and more cell death in response to extended GD injury versus astrocytes from the hippocampal DG subregion. This functional, subregional heterogeneity is neutralized by exogenously elevating miR-29a levels, or by siRNA-mediated knockdown of the miR-29a target VDAC1. Moreover, blocking the miR-29a/VDAC1 interaction results in augmented expression of VDAC1, resulting in an increase in pre-injury levels
Acknowledgments
Supported by American Heart Association14FTF-19970029 to Dr. Stary and by National Institutes of Health grants R01 NS084396, R01 NS053898 and R01 NS 080177 to Dr. Giffard.
References (38)
- et al.
MicroRNA-7 regulates the function of mitochondrial permeability transition pore by targeting VDAC1 expression
J. Biol. Chem.
(2016) - et al.
Voltage-dependent anion channels (VDACs) promote mitophagy to protect neuron from death in an early brain injury following a subarachnoid hemorrhage in rats
Brain Res.
(2014) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method
Methods
(2001) - et al.
The role of VDAC in cell death: friend or foe?
Biochim. Biophys. Acta
(2012) Epigenetic principles and mechanisms underlying nervous system functions in health and disease
Prog. Neurobiol.
(2008)- et al.
Overexpression of inducible heat shock protein 70 and its mutants in astrocytes is associated with maintenance of mitochondrial physiology during glucose deprivation stress
Cell Stress Chaperones
(2006) - et al.
Overexpressing GRP78 influences Ca2 + handling and function of mitochondria in astrocytes after ischemia-like stress
Mitochondrion
(2011) - et al.
Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes
Brain Res.
(1997) - et al.
MicroRNAs: novel therapeutic targets in neurodegenerative diseases
Drug Discov. Today
(2009) - et al.
VDAC, a multi-functional mitochondrial protein as a pharmacological target
Mitochondrion
(2012)
Mitochondria, oxidative metabolism and cell death in stroke
Biochim. Biophys. Acta
HSP70 protects murine astrocytes from glucose deprivation injury
Neurosci. Lett.
Differential sensitivity of murine astrocytes and neurons from different brain regions to injury
Exp. Neurol.
Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death
Nat. Cell Biol.
Identification of novel targets for miR-29a using miRNA proteomics
PLoS One
Astrocytes: targets for neuroprotection in stroke
Cent. Nerv. Syst. Agents Med. Chem.
Emerging roles of astrocytes in neural circuit development
Nat. Rev. Neurosci.
Dissection of hippocampal dentate gyrus from adult mouse
J. Vis. Exp.
Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2 + uptake and reactive oxygen species generation
Cell Death Dis.
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Primary authors.