Nishani T. Hettiarachchi

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson’s disease (PD) is characterized in part by the presence of α‐synuclein (α‐syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α‐synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura‐2 loaded SH‐SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild‐type α‐syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage‐gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of α‐syn, an effect which was due specifically to increased Ca2+ entry via L‐type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the α‐syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of α‐synuclein. However, only WT α‐syn transfected cells displayed significantly impaired viability. Our findings suggest that α‐syn regulates Ca2+ entry pathways and, consequently, that abnormal α‐syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Resistance of dynamin-related protein 1 oligomers to disassembly impairs mitophagy, resulting in myocardial inflammation and heart failure.

Background: The C452F mutation in the mitochondrial fission protein Drp1 leads to heart failure through an unknown mechanism. Results: C452F impairs Drp1 disassembly, leading to impaired mitophagy, failed bioenergetics, and inflammation. Conclusion: Drp1-mediated mitochondrial fission is essential for normal cardiac function. Significance: Mutations in mitochondrial quality control proteins are a likely cause of human cardiomyopathy. We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Heme oxygenase-1 protects against Alzheimer’s amyloid- β 1-42 -induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer’s disease, catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediates cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentration-dependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc-expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.

Peroxynitrite Mediates Disruption of Ca2+ Homeostasis by Carbon Monoxide via Ca2+ ATPase Degradation

AIM Sublethal carbon monoxide poisoning causes prolonged neurological damage involving oxidative stress. Given the central role of Ca(2+) homeostasis and its vulnerability to stress, we investigated whether CO disrupts neuronal Ca(2+) homeostasis. RESULTS Cytosolic Ca(2+) transients evoked by muscarine in SH-SY5Y cells were prolonged by CO (applied via the donor CORM-2), and capacitative Ca(2+) entry (CCE) was dramatically enhanced. Ca(2+) store mobilization by cyclopiazonic acid was similarly augmented, as was the subsequent CCE, and that evoked by thapsigargin. Ca(2+) rises evoked by depolarization were also enhanced by CO, and Ca(2+) levels often did not recover in its presence. CO increased intracellular nitric oxide (NO) and all effects of CO were prevented by inhibiting NO formation. However, NO donors did not mimic the effects of CO. The antioxidant ascorbic acid inhibited effects of CO on Ca(2+) signaling, as did the peroxynitrite scavenger, FeTPPS, and CO increased peroxynitrite formation. Finally, CO caused significant loss of plasma membrane Ca(2+)ATPase (PMCA) protein, detected by Western blot, and this was also observed in brain tissue of rats exposed to CO in vivo. INNOVATION The cellular basis of CO-induced neurotoxicity is currently unknown. Our findings provide the first data to suggest signaling pathways through which CO causes neurological damage, thereby opening up potential targets for therapeutic intervention. CONCLUSION CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.

Heme oxygenase-1 derived carbon monoxide suppresses Aβ1–42 toxicity in astrocytes

Neurodegeneration in Alzheimer’s disease (AD) is extensively studied, and the involvement of astrocytes and other cell types in this process has been described. However, the responses of astrocytes themselves to amyloid β peptides ((Aβ; the widely accepted major toxic factor in AD) is less well understood. Here, we show that Aβ(1-42) is toxic to primary cultures of astrocytes. Toxicity does not involve disruption of astrocyte Ca2+ homeostasis, but instead occurs via formation of the toxic reactive species, peroxynitrite. Thus, Aβ(1-42) raises peroxynitrite levels in astrocytes, and Aβ(1-42) toxicity can be inhibited by antioxidants, or by inhibition of nitric oxide (NO) formation (reactive oxygen species (ROS) and NO combine to form peroxynitrite), or by a scavenger of peroxynitrite. Increased ROS levels observed following Aβ(1-42) application were derived from NADPH oxidase. Induction of haem oxygenase-1 (HO-1) protected astrocytes from Aβ(1-42) toxicity, and this protective effect was mimicked by application of the carbon monoxide (CO) releasing molecule CORM-2, suggesting HO-1 protection was attributable to its formation of CO. CO suppressed the rise of NADPH oxidase-derived ROS caused by Aβ(1-42). Under hypoxic conditions (0.5% O2, 48 h) HO-1 was induced in astrocytes and Aβ(1-42) toxicity was significantly reduced, an effect which was reversed by the specific HO-1 inhibitor, QC-15. Our data suggest that Aβ(1-42) is toxic to astrocytes, but that induction of HO-1 affords protection against this toxicity due to formation of CO. HO-1 induction, or CO donors, would appear to present attractive possible approaches to provide protection of both neuronal and non-neuronal cell types from the degenerative effects of AD in the central nervous system.

Cellular consequences of the expression of Alzheimer's disease-causing presenilin 1 mutations in human neuroblastoma (SH-SY5Y) cells

Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimers disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca2+ concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca2+ -free solutions did not deplete Ca2+ stores, demonstrating there was no difference in Ca2+ leak from endoplasmic reticulum (ER) stores in PS1 mutants. Peak muscarine-evoked rises of [Ca2+]i were variable, but the integrals were not significantly different, suggesting, while kinetics of Ca2+ store release might be affected in PS1 mutants, store size was similar. However, when Ca2+ -ATPase activity was irreversibly inhibited with thapsigargin, the S170F and ΔE9 cells showed larger capacitative calcium entry indicating a direct effect on Ca2+ influx pathways. There was no significant effect of any of the mutations on mitochondrial respiration. Amyloid β(Aβ(1-40)) secretion was reduced, and Aβ(1-42) secretion increased in the S170F cells resulting in a very large increase in the Aβ42/40 ratio. This, rather than any potential disruption of ER Ca2+ stores, is likely to explain the extreme pathology of this mutant.

Hypoxic remodelling of Ca2+ stores does not alter human cardiac myofibroblast invasion

Cardiac fibroblasts are the most abundant cell type in the heart, and play a key role in the maintenance and repair of the myocardium following damage such as myocardial infarction by transforming into a cardiac myofibroblast (CMF) phenotype. Repair occurs through controlled proliferation and migration, which are Ca(2+) dependent processes, and often requires the cells to operate within a hypoxic environment. Angiotensin converting enzyme (ACE) inhibitors reduce infarct size through the promotion of bradykinin (BK) stability. Although CMF express BK receptors, their activity under the reduced O(2) conditions that occur following infarct are entirely unexplored. Using Fura-2 microfluorimetry on primary human CMF, we found that hypoxia significantly increased the mobilisation of Ca(2+) from intracellular stores in response to BK whilst capacitative Ca(2+) entry (CCE) remained unchanged. The enhanced store mobilisation was due to a striking increase in CMF intracellular Ca(2+)-store content under hypoxic conditions. However, BK-induced CMF migration or proliferation was not affected following hypoxic exposure, suggesting that Ca(2+) influx rather than mobilisation is of primary importance in CMF migration and proliferation.

Gap junction-mediated spontaneous Ca2+ waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

Hypoxic remodelling of Ca2+signalling in SH-SY5Y cells: influence of glutathione.

Prolonged hypoxia alters various cellular processes, including Ca2+ signalling. As these effects can be prevented by antioxidants, we examined the role of glutathione, the major intracellular redox buffer, in modulation of Ca2+ signalling in the human neuroblastoma SH-SY5Y by hypoxia. Rises of [Ca2+]i evoked by bradykinin, and subsequent capacitative Ca2+ entry, were enhanced by prior hypoxia (1% O2, 24 h) without effect on reduced glutathione levels. Glutathione depletion reversed the effects of chronic hypoxia, but did not affect normoxically cultured cells. Elevation of glutathione levels also prevented the effects of hypoxia, but restored such effects in glutathione-depleted cells. Glutathione is therefore required for hypoxia to modify Ca2+ signalling, but its role is more complex than simple buffering of reactive oxygen species.

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO‐induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole‐cell current‐ and voltage‐clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild‐type or a C723S mutant form of ether‐a‐go‐go–related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO−) in HEK293 cells fluorimetrically. CO—applied as the CO‐releasing molecule, CORM‐2—prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild‐type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO− levels, an effect that was reversed by the ONOO− scavenger, FeTPPS [5,10,15,20‐tetrakis‐(4‐sulfonatophenyl)‐porphyrinato‐iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO−‐mediated inhibition of Kv11.1 K+ channels.—Al‐Owais, M.M., Hettiarachchi, N.T., Kirton, H.M., Hardy, M.E., Boyle, J.P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite‐mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations. FASEB J. 31, 4845–4854 (2017). www.fasebj.org