Birgit Honrath

Inhibition of HIF-prolyl-4-hydroxylases prevents mitochondrial impairment and cell death in a model of neuronal oxytosis

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

Activation of SK2 channels preserves ER Ca(2+) homeostasis and protects against ER stress-induced cell death

Alteration of endoplasmic reticulum (ER) Ca2+ homeostasis leads to excessive cytosolic Ca2+ accumulation and delayed neuronal cell death in acute and chronic neurodegenerative disorders. While our recent studies established a protective role for SK channels against excessive intracellular Ca2+ accumulation, their functional role in the ER has not been elucidated yet. We show here that SK2 channels are present in ER membranes of neuronal HT-22 cells, and that positive pharmacological modulation of SK2 channels with CyPPA protects against cell death induced by the ER stressors brefeldin A and tunicamycin. Calcium imaging of HT-22 neurons revealed that elevated cytosolic Ca2+ levels and decreased ER Ca2+ load during sustained ER stress could be largely prevented by SK2 channel activation. Interestingly, SK2 channel activation reduced the amount of the unfolded protein response transcription factor ATF4, but further enhanced the induction of CHOP. Using siRNA approaches we confirmed a detrimental role for ATF4 in ER stress, whereas CHOP regulation was dispensable for both, brefeldin A toxicity and CyPPA-mediated protection. Cell death induced by blocking Ca2+ influx into the ER with the SERCA inhibitor thapsigargin was not prevented by CyPPA. Blocking the K+ efflux via K+/H+ exchangers with quinine inhibited CyPPA-mediated neuroprotection, suggesting an essential role of proton uptake and K+ release in the SK channel-mediated neuroprotection. Our data demonstrate that ER SK2 channel activation preserves ER Ca2+ uptake and retention which determines cell survival in conditions where sustained ER stress contributes to progressive neuronal death.

The role of Ca(2+) in cell death caused by oxidative glutamate toxicity and ferroptosis

Ca2+ ions play a fundamental role in cell death mediated by oxidative glutamate toxicity or oxytosis, a form of programmed cell death similar and possibly identical to other forms of cell death like ferroptosis. Ca2+ influx from the extracellular space occurs late in a cascade characterized by depletion of the intracellular antioxidant glutathione, increases in cytosolic reactive oxygen species and mitochondrial dysfunction. Here, we aim to compare oxidative glutamate toxicity with ferroptosis, address the signaling pathways that culminate in Ca2+ influx and cell death and discuss the proteins that mediate this. Recent evidence hints toward a role of the machinery responsible for store-operated Ca2+ entry (SOCE), which refills the endoplasmic reticulum (ER) after receptor-mediated ER Ca2+ release or other forms of store depletion. Pharmacological inhibition of SOCE or transcriptional downregulation of proteins involved in SOCE like the ER Ca2+ sensor STIM1, the plasma membrane Ca2+ channels Orai1 and TRPC1 and the linking protein Homer protects against oxidative glutamate toxicity and direct oxidative stress caused by hydrogen peroxide or 1-methyl-4-phenylpyridinium (MPP+) injury, a cellular model of Parkinsons disease. This suggests that SOCE inhibition might have some potential therapeutic effects in human disease associated with oxidative stress like neurodegenerative disorders.

Glucose-regulated protein 75 determines ER–mitochondrial coupling and sensitivity to oxidative stress in neuronal cells

The crosstalk between different organelles allows for the exchange of proteins, lipids and ions. Endoplasmic reticulum (ER) and mitochondria are physically linked and signal through the mitochondria-associated membrane (MAM) to regulate the transfer of Ca2+ from ER stores into the mitochondrial matrix, thereby affecting mitochondrial function and intracellular Ca2+ homeostasis. The chaperone glucose-regulated protein 75 (GRP75) is a key protein expressed at the MAM interface which regulates ER–mitochondrial Ca2+ transfer. Previous studies revealed that modulation of GRP75 expression largely affected mitochondrial integrity and vulnerability to cell death. In the present study, we show that genetic ablation of GRP75, by weakening ER–mitochondrial junctions, provided protection against mitochondrial dysfunction and cell death in a model of glutamate-induced oxidative stress. Interestingly, GRP75 silencing attenuated both cytosolic and mitochondrial Ca2+ overload in conditions of oxidative stress, blocked the formation of reactive oxygen species and preserved mitochondrial respiration. These data revealed a major role for GRP75 in regulating mitochondrial function, Ca2+ and redox homeostasis. In line, GRP75 overexpression enhanced oxidative cell death induced by glutamate. Overall, our findings suggest weakening ER–mitochondrial connectivity by GRP75 inhibition as a novel protective approach in paradigms of oxidative stress in neuronal cells.

Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways

Ca2+-activated K+ channels (KCa) are expressed at the plasma membrane and in cellular organelles. Expression of all KCa channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial KCa channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial KCa channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart.

One protein, different cell fate: the differential outcome of depleting GRP75 during oxidative stress in neurons

The interconnection between the endoplasmic reticulum (ER) and mitochondria to transfer Ca into the mitochondrial matrix constitutes a major part of intracellular Ca signaling. By increasing mitochondrial Ca ([Ca]m) uptake, ER-mitochondrial crosstalk enhances energy production through accelerating mitochondrial respiration, thereby supporting cellular function and survival. ER-mitochondrial associations are established by multiprotein complexes formed, for instance, by ER-bound inositol-1,4,5-trisphosphate receptor (IP3R), mitochondriaresident voltage-dependent anion channel 1 (VDAC1) and the heat shock protein glucose-regulated protein 75 (GRP75). While IP3R and VDAC1 are Ca -permeable ion channels driving Ca flux, GRP75 is essential to maintain the physical contact between the organelles, thereby facilitating the propagation of the Ca signal into the mitochondria. Reduced GRP75 expression in tumor cells derived from bone, breast or colon has been linked to an increased susceptibility to cell death, and small molecule GRP75-inhibitory drugs are exploited as a potential therapeutic intervention. However, the relevance of GRP75 for ER-mitochondrial crosstalk in neurons or brain-derived tumor cells is largely unknown. Under physiological conditions, GRP75 inhibition seemed to activate mitochondrial stress responses such as the mitochondrial unfolded protein response or autophagy in human neuroblastoma SH-SY5Y cells. In contrast, under pathological conditions, GRP75 expression exerted different effects. For instance, in SH-SY5Y cells increased GRP75 expression prevented mitochondrial dysfunction and cell death following proteolytic stress induced by overexpression of mitochondrial ornithine transcarbamylase. In contrast, in human dopaminergic neurons, GRP75 overexpression potentiated the cytotoxic effects of the mitochondrial complex I inhibitor rotenone. Our recent study published in Cell Death & Discovery provided further insights into the role of GRP75 on mitochondrial dysfunction and cell death in a neuronal model of oxidative stress. Using immortalized hippocampal HT22 cells, we investigated the consequences of GRP75 expression on cell survival in a model of glutamate-induced oxytosis. In this study, inhibition and/ or gene silencing of GRP75 via siRNA or CRISPR/Cas9knockout fully prevented the oxidative cell death. In particular, GRP75 depletion exerted protection by preserving the mitochondrial network, preventing mitochondrial membrane depolarization and restoring the mitochondrial redox balance. Furthermore, GRP75 depletion restored Ca homeostasis by preventing [Ca]m overload and late-stage cytosolic Ca 2+ dysregulation mediated by Ca release-activated calcium channel protein 1 (ORAI1). In turn, elevating GRP75 expression increased the sensitivity of neural HT22 cells towards the glutamate challenge. Notably, neither pharmacological inhibition alone nor genetic downregulation of GRP75 had any effect on cell survival or mitochondrial function in control conditions. GRP75 gene silencing neither impaired cell proliferation, nor altered the mitochondrial network, mitochondrial ROS, or the mitochondrial membrane potential. We therefore suggest that the GRP75-dependent ER-mitochondrial coupling is a major determinant of cell fate in conditions of oxidative stress, without affecting

Small conductance Ca2+-activated K+ channels in the plasma membrane, mitochondria and the ER : Pharmacology and implications in neuronal diseases

Ca2+-activated K+ (KCa) channels regulate after-hyperpolarization in many types of neurons in the central and peripheral nervous system. Small conductance Ca2+-activated K+ (KCa2/SK) channels, a subfamily of KCa channels, are widely expressed in the nervous system, and in the cardiovascular system. Voltage-independent SK channels are activated by alterations in intracellular Ca2+ ([Ca2+]i) which facilitates the opening of these channels through binding of Ca2+ to calmodulin that is constitutively bound to the SK2 C-terminus. In neurons, SK channels regulate synaptic plasticity and [Ca2+]i homeostasis, and a number of recent studies elaborated on the emerging neuroprotective potential of SK channel activation in conditions of excitotoxicity and cerebral ischemia, as well as endoplasmic reticulum (ER) stress and oxidative cell death. Recently, SK channels were discovered in the inner mitochondrial membrane and in the membrane of the endoplasmic reticulum which sheds new light on the underlying molecular mechanisms and pathways involved in SK channel-mediated protective effects. In this review, we will discuss the protective properties of pharmacological SK channel modulation with particular emphasis on intracellularly located SK channels as potential therapeutic targets in paradigms of neuronal dysfunction.

SK channel activation is neuroprotective in conditions of enhanced ER-mitochondrial coupling

Alterations in the strength and interface area of contact sites between the endoplasmic reticulum (ER) and mitochondria contribute to calcium (Ca2+) dysregulation and neuronal cell death, and have been implicated in the pathology of several neurodegenerative diseases. Weakening this physical linkage may reduce Ca2+ uptake into mitochondria, while fortifying these organelle contact sites may promote mitochondrial Ca2+ overload and cell death. Small conductance Ca2+-activated K+ (SK) channels regulate mitochondrial respiration, and their activation attenuates mitochondrial damage in paradigms of oxidative stress. In the present study, we enhanced ER–mitochondrial coupling and investigated the impact of SK channels on survival of neuronal HT22 cells in conditions of oxidative stress. Using genetically encoded linkers, we show that mitochondrial respiration and the vulnerability of neuronal cells to oxidative stress was inversely linked to the strength of ER–mitochondrial contact points and the increase in mitochondrial Ca2+ uptake. Pharmacological activation of SK channels provided protection against glutamate-induced cell death and also in conditions of increased ER–mitochondrial coupling. Together, this study revealed that SK channel activation provided persistent neuroprotection in the paradigm of glutamate-induced oxytosis even in conditions where an increase in ER–mitochondrial coupling potentiated mitochondrial Ca2+ influx and impaired mitochondrial bioenergetics.