Elsevier

Mitochondrion

Volume 30, September 2016, Pages 248-254
Mitochondrion

miR-29a differentially regulates cell survival in astrocytes from cornu ammonis 1 and dentate gyrus by targeting VDAC1

https://doi.org/10.1016/j.mito.2016.08.013Get rights and content

Highlights

  • Hippocampal CA1 astrocytes exhibit lower miR-29a levels and greater injury from glucose deprivation than DG astrocytes.

  • Elevations in VDAC1, a miR-29a target, coincide with increased cell death, greater in CA1 astrocytes.

  • CA1/DG differences in susceptibility to injury are neutralized by elevating miR-29a, or by knockdown of VDAC1.

  • Blocking the miR-29a/VDAC1 interaction augments expression of VDAC1, reducing miR-29a protection.

  • These findings suggest that VDAC1 plays a downstream role in the miR-29a response to injury in astrocytes.

Abstract

Neurons in the cornu ammonis 1 (CA1) region of the hippocampus are vulnerable to cerebral ischemia, while dentate gyrus (DG) neurons are more resistant. This effect is mediated by local astrocytes, and may reflect differences in subregional hippocampal expression of miR-29a. We investigated the role of miR-29a on survival of hippocampal astrocytes cultured selectively from CA1 and DG in response to glucose deprivation (GD). CA1 astrocytes exhibited more cell death and a greater decrease in miR-29a than DG astrocytes. A reciprocal change was observed in the mitochondrial voltage dependent cation channel-1 (VDAC1), a regulator of mitochondria and target of miR-29a. In CA1 astrocytes, increasing miR-29a decreased VDAC1 and improved cell survival, while knockdown of VDAC1 improved survival. Finally, the protective effect of miR-29a was eliminated by inhibition of miR-29a/VDAC1 binding. These findings suggest that the selective vulnerability of the CA1 to injury may be due in part to a limited miR-29a response in CA1 astrocytes, allowing a greater increase in VDAC1-mediated cellular dysfunction in CA1 astrocytes.

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)

  • N.R. Sims et al.

    Mitochondria, oxidative metabolism and cell death in stroke

    Biochim. Biophys. Acta

    (2010)
  • L. Xu et al.

    HSP70 protects murine astrocytes from glucose deprivation injury

    Neurosci. Lett.

    (1997)
  • L. Xu et al.

    Differential sensitivity of murine astrocytes and neurons from different brain regions to injury

    Exp. Neurol.

    (2001)
  • C.P. Baines et al.

    Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death

    Nat. Cell Biol.

    (2007)
  • R. Bargaje et al.

    Identification of novel targets for miR-29a using miRNA proteomics

    PLoS One

    (2012)
  • G. Barreto et al.

    Astrocytes: targets for neuroprotection in stroke

    Cent. Nerv. Syst. Agents Med. Chem.

    (2011)
  • L.E. Clarke et al.

    Emerging roles of astrocytes in neural circuit development

    Nat. Rev. Neurosci.

    (2013)
  • H. Hagihara et al.

    Dissection of hippocampal dentate gyrus from adult mouse

    J. Vis. Exp.

    (2009)
  • H. Huang et al.

    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.

    (2014)
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    Primary authors.

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