Role of hypoxia-inducible factor-prolyl-hydroxylase (HIF-PHD) in mitochondrial pathways of ischemic neuronal death

• Applicant: Professor Dr. Carsten Culmsee
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: from 2018 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 391520971

• Final Report Year: 2022

Final Report Abstract

Hypoxia-inducible factor (HIF) prolyl-4-hydroxylases (PHDs) were revealed as promising target candidates for mitochondrial protection in paradigms of oxidative stress associated with ferroptosis and necroptosis signaling in vitro and in vivo. In the present DFG project we revealed that inhibitors of HIF-PHDs provide protective effects in paradigms of regulated necrosis and in models of hypoxic/ischemic brain damage. Most importantly, combining the expertise of the applicants in this project allowed for achieving a unique and unifying picture on the role of mitochondria in the cell death paradigms of ferroptosis and necroptosis, and on the role of HIF-PHD and associated bioenergetic regulation and, in particular we identified the new integrating key regulator CYLD in these HIF-PHD- dependent death signaling pathways relevant to acute brain injury. We identified inhibition of mitochondrial respiration and a metabolic shift to glycolysis as a general mechanism imposed by HIF-PHD inhibition, CYLD depletion or by MitoQ to preserve mitochondrial integrity and prevent excessive mitochondrial ROS production in neural cells exposed to ferroptosis stimuli. Based on these findings, we conclude that reduced mitochondrial respiration prevented neural cells from detrimental mitochondrial ROS formation and cell death, while the required energy supply was delivered through enhanced glycolysis. Further, we identified a pivotal integrating role for CYLD regulating HIF-PHD and HIF-dependent bioenergetic activities at the level of mitochondria and driving RIP1 activation and necrosome formation in ferroptotic death pathways in neurons; and these mechanisms are apparently highly relevant for mechanisms of brain damage after cerebral ischemia and brain trauma as demonstrated by our findings using the HIF-PHD inhibitor adaptaquin in the model of neonatal hypoxia/ischemia and in CYLD ko mice in models of transient focal ischemia and brain trauma. Finally, we also evaluated the role of PHDs inhibition in the regulation of neuroinflammatory responses in cultured microglial cells stimulated by LPS and showed that microglial activity may also contribute to neuroprotection in organotypic slice cultures. These preliminary data are also partly confirming our previous hypotheses and need further experimental work for validation and publication. Overall, the combined and collaborative investigations in both laboratories provide evidence that manipulation of ferroptosis and necroptosis signaling at the level of HIF-PHD-dependent regulation of mitochondrial bioenergetic activities are important for cellular resilience in neural cells and in brain tissue. Despite the ongoing restrictions for laboratory work by the Covid-19 pandemic, the detrimental flood in Zhengzhou 2021 and ongoing travel restrictions the collaborative work between the partners of the joined NSFC/DFG project was fruitful and highly successful and is also documented in joined publications. The exchange between the laboratories was stimulated by frequent online meetings and, before pandemic restrictions, also in personal meetings. Afterwards, traveling was restricted and further restrictions and lockdown activities, and finally the flood catastrophe in Zhengzhou slowed down collaborative work but not online-meetings and successful experimental work and publication activities that are still ongoing.

Publications

• Cylindromatosis mediates neuronal cell death in vitro and in vivo. Cell Death Differ. 2018; 25: 1394-1407
  Ganjam GK, Terpolilli NA, Diemert S, Eisenbach I, Hoffmann L, Reuther C, Herden C, Roth J, Plesnila N, Culmsee C
  
• Mitochondrial rescue prevents glutathione peroxidase-dependent ferroptosis. Free Radic Biol Med. 2018; 117: 45-57
  Jelinek A, Heyder L, Daude M, Plessner M, Krippner S, Grosse R, Diederich WE, Culmsee C
  
• Actin(g) on mitochondria - a role for cofilin1 in neuronal cell death pathways. Biol Chem. 2019;400: 1089-1097
  Hoffmann L, Rust MB, Culmsee C
  
• Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons. Cell Death Dis. 2019; 10: 865
  Ganjam GK, Bolte K, Matschke LA, Neitemeier S, Dolga AM, Höllerhage M, Höglinger GU, Adamczyk A, Decher N, Oertel WH, Culmsee C
  
• Sex differences in neonatal mouse brain injury after hypoxia-ischemia and adaptaquin treatment, Journal of Neurochemistry, 2019, 150: 759-775
  Li, Kenan; Li, Tao; Wang, Yafeng; Xu, Yiran; Zhang, Shan; Culmsee, Carsten, Wang, Xiaoyang; Zhu, Changlian
  
• The Potential Role of Ferroptosis in Neonatal Brain Injury, Frontiers in Neuroscience, 2019, 13: 115
  Wu, Yanan; Song, Juan; Wang, Yafeng; Wang, Xiaoyang; Culmsee, Carsten, Zhu, Changlian
  
• Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration. Cells. 2020; 9: 2259
  Clemente LP, Rabenau M, Tang S, Stanka J, Cors E, Stroh J, Culmsee C, von Karstedt S
  
• The neuroprotective role of microglial cells against amyloid beta-mediated toxicity in organotypic hippocampal slice cultures. Brain Pathol. 2020, 30: 589-602
  Richter M, Vidovic N, Biber K, Dolga A, Culmsee C, Dodel R
  
• Interaction between BID and VDAC1 is required for mitochondrial demise and cell death in neurons. bioRxiv
  Sina Oppermann, Barbara Mertins, Lilja Meissner, Cornelius Krasel, Georgios Psakis, Philipp Reiß, Amalia M. Dolga, Nikolaus Plesnila, Moritz Bünemann, Lars-Oliver Essen, Carsten Culmsee
  
• RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury. Acta Neuropathol Commun. 2021; 9: 138
  Wehn AC, Khalin I, Duering M, Hellal F, Culmsee C, Vandenabeele P, Plesnila N, Terpolilli NA