Original research articleHypoxia-reoxygenation of primary astrocytes results in a redistribution of mitochondrial size and mitophagy
Introduction
Stroke is a cerebrovascular event characterized by severe cerebral ischemia that is the fifth leading cause of death and is the primary contributor of adult disability in the U.S. (Kochanek et al. 2014; Mozaffarian et al. 2015). During stroke there is a cerebral cellular energy crisis caused by a decline in the delivery of the substrates, glucose and molecular oxygen, resulting in a compromised synthesis of ATP through a collapse of oxidative phosphorylation and glycolysis (Rossi et al. 2007). Insufficient cellular ATP content interrupts a wide range of indispensable ATP-dependent processes, including ion balances across neuronal membranes (Hansen and Nedergaard 1988; Silver et al. 1997). An elevation of free cytosolic Ca2+ levels via voltage-gated and receptor-gated calcium channels is the central effector initiating the massive release of extracellular glutamate, and the primary cause of excitotoxicity in ischemia (Katayama et al. 1991; Duffy and MacVicar 1996; Parpura and Haydon 2000). Clearance of synaptic glutamate is a core function of astrocytes that helps protect against glutamate toxicity (Rothstein et al. 1996).
Also, astrocytes are critical for providing neurons with a source of glutamine necessary for glutamate production through a process known as the glutamate-glutamine cycle (Waniewski and Martin, 1986; Chaudhry et al. 2002). Embargo of glutamine delivery to neurons by the blockade of astrocytic conversion of glutamate to glutamine has been reported to reduce the potassium-evoked glutamate release in experimental models of focal ischemia, reducing infarct size (Paulsen and Fonnum 1989; Swanson et al. 1990). Astrocytic mitochondria are key organelles that allow astrocytes to participate in such extensive metabolic activities (Nehlig et al. 2004; Lovatt et al. 2007).
Here we investigated the effects of hypoxia and post-hypoxia reoxygenation on astrocytic mitochondrial structure, including mitochondrial dimensions and content, as well as the underlying mechanism(s) and functions of these dynamic changes. This research provides evidence of early mitochondrial fission during hypoxia-reoxygenation that may participate in the damaging effects of ischemic insult to the central nervous system.
Section snippets
Preparation of primary astrocytic cultures
Primary astrocytic cultures were prepared using a method previously reported, which generated an 85% yield of astrocytes, and a 15%, and < 1% yield of progenitor cells and microglia, respectively (Almeida and Medina 1998). Embryonic day 19 pregnant rats were deeply anesthetized with isoflurane. After confirming deep anesthetization via tail pinch, rats received a 3-in. vertical incision to the lower abdomen. Once the incision was made, the embryos including the placenta and amniotic sacks were
Mitochondrial size measurements
Astrocytic exposure to hypoxia demonstrated the dynamic ability of mitochondria to undergo fission and fusion in response to changes in environmental oxygen pressure (Fig. 1A and B). Hypoxic exposure for 3-hours with or without 10-hours reoxygenation resulted in a redistribution of mitochondrial size to a larger number of smaller mitochondria.
Primary astrocytes that were exposed to 3-hours hypoxia with no reoxygenation showed the largest redistribution to smaller sized mitochondria compared to
Discussion
In this study, we have demonstrated that hypoxia and post-hypoxia reoxygenation of primary astrocytes results in a redistribution of mitochondria to smaller sizes evoked by an increase in mitochondrial fission. Excessive mitochondrial fission corresponded to Drp-1 dephosphorylation at Ser 637, which preceded mitophagy of relatively small mitochondria. Post-hypoxia reoxygenation of astrocytes marked the initiation of elevated mitophagic activity particularly in the perinuclear region where a
Author contribution statement
DDQ designed studies, conducted studies and composed the manuscript. JAG, SNS, SJ, EBE-C, AER, and JZC aided with studies, analyzed data, and revised the manuscript. JWS designed studies and revised the manuscript.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This work was supported by NIH grants P20 GM109098, P01 AG027956, U54 GM104942 and T32 AG052375. Imaging experiments and image analysis were performed in the West Virginia University Microscope Imaging Facility, which has been supported by the Mary Babb Randolph Cancer Center and NIH grants T32 AG052375, P20 RR016440, P30 RR032138/GM103488 and P20 RR016477.
References (70)
- et al.
A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture
Brain Res. Protocol.
(1998) - et al.
Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO
FEBS Lett.
(1987) - et al.
Mitochondria, oxidants, and aging
Cell
(2005) - et al.
Stimulation of glutamate receptors in cultured hippocampal neurons causes Ca2+−dependent mitochondrial contraction
Cell Calcium
(2009) - et al.
Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology
J. Biol. Chem.
(2007) - et al.
Mitochondria supply membranes for autophagosome biogenesis during starvation
Cell
(2010) - et al.
Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells
Biochem. Biophys. Res. Commun.
(2003) - et al.
Swelling of mitochondria in cultured rat hippocampal astrocytes is induced by high cytosolic Ca2+ load, but not by mitochondrial depolarization
FEBS Lett.
(2002) - et al.
Calcium-dependent glutamate release concomitant with massive potassium flux during cerebral ischemia in vivo
Brain Res.
(1991) Increased release of excitatory amino acids by the actions of ATP and peroxynitrite on volume-regulated anion channels (VRACs) in astrocytes
Neurochem. Int.
(2004)