Ischemia-induced autophagy contributes to neurodegeneration in cerebellar Purkinje cells in the developing rat brain and in primary cortical neurons in vitro

Alicia K Au; Yaming Chen; Lina Du; Craig M Smith; Mioara D Manole; Sirine A Baltagi; Charleen T Chu; Rajesh K Aneja; Hülya Bayır; Patrick M Kochanek; Robert S B Clark

doi:10.1016/j.bbadis.2015.06.007

Ischemia-induced autophagy contributes to neurodegeneration in cerebellar Purkinje cells in the developing rat brain and in primary cortical neurons in vitro

Biochim Biophys Acta. 2015 Sep;1852(9):1902-11. doi: 10.1016/j.bbadis.2015.06.007. Epub 2015 Jun 11.

Authors

Affiliations

¹ Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
² Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
³ Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.
⁴ Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
⁵ Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
⁶ Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.
⁷ Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
⁸ Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA; Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
⁹ Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA; Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address: clarkrs@upmc.edu.

Abstract

Increased autophagy/mitophagy is thought to contribute to cerebellar dysfunction in Purkinje cell degeneration mice. Intriguingly, cerebellar Purkinje cells are highly vulnerable to hypoxia-ischemia (HI), related at least in part to their high metabolic activity. Whether or not excessive or supraphysiologic autophagy plays a role in Purkinje cell susceptibility to HI is unknown. Accordingly, we evaluated the role of autophagy in the cerebellum after global ischemia produced by asphyxial cardiac arrest in postnatal day (PND) 16-18 rats, using siRNA-targeted inhibition of Atg7, necessary for microtubule-associated protein light chain 3-II (LC3-II) and Atg12-Atg5 complex formation. Two days before a 9min asphyxial cardiac arrest or sham surgery, Atg7 or control siRNA was injected intracisternally to target the cerebellum. Treatment with Atg7 siRNA: 1) reduced Atg7 protein expression in the cerebellum by 56%; 2) prevented the typical ischemia-induced formation of LC3-II in the cerebellum 24h after asphyxial cardiac arrest; 3) improved performance on the beam-balance apparatus on days 1-5; and 4) increased calbindin-labeled Purkinje cell survival assessed on day 14. Improved Purkinje cell survival was more consistent in female vs. male rats, and improved beam-balance performance was only seen in female rats. Similar responses to Atg7 siRNA i.e. reduced autophagy and neurodegeneration vs. control siRNA were seen when exposing sex-segregated green fluorescent protein-LC3 tagged mouse primary cortical neurons to oxygen glucose deprivation in vitro. Thus, inhibition of autophagy after global ischemia in PND 16-18 rats leads to increased survival of Purkinje cells and improved motor performance in a sex-dependent manner.

Keywords: Asphyxia; Atg7; Cardiac arrest; Cerebellum; Hypoxia–ischemia; Ischemic brain injury; Oxygen glucose deprivation; Purkinje neuron; Small interfering RNA.

Abstract

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