High-Resolution Respirometry in Assessment of Mitochondrial Function in Neuroblastoma SH-SY5Y Intact Cells

Evinová, Andrea; Čižmárová, Beáta; Hatokova, Zuzana; Račay, Peter

doi:10.1007/s00232-020-00107-4

Cited by 12 publications

(9 citation statements)

References 29 publications

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“…Moreover, we also found that MMA reduced cellular ATP content (Figure 5B), which are in agreement with the reduction observed in ATP-linked respiration (Figure 3B). More importantly, reserve respiratory capacity, which describes the extra ATP that can be produced by mitochondria in response, for example, to ATP-dependent pump activation to maintain ionic potentials and neurotransmitter uptake (Evinova et al 2020), was also reduced by all MMA tested concentrations (Figure 3D), reinforcing the relationship between mitochondrial dysfunction and reduced glutamate uptake elicited by MMA in our experimental model. Moreover, there is accumulating evidence that inability of astrocytes to deal with increased glutamate concentrations can lead to neuronal death though excitotoxic mechanisms (Kauppinen, Swanson 2007;Gerkau et al 2017).…”

Section: Discussionsupporting

confidence: 65%

“…Routine and ATP-linked respiration are related to oxygen consumption employed to supply cellular regular metabolism at basal state, being dependent on substrate oxidation, ATP turnover and proton leak (Brand, Nicholls 2011). Maximal respiration reflects the maximal electron flux in the electron transfer chain and reserve respiratory capacity describes the amount of extra ATP that can be produced by mitochondria in response to a rapid increase in energy demand, like during ATP-dependent pump activation to maintain ionic potentials and neurotransmitter uptake (Evinova et al 2020).…”

Section: Discussionmentioning

confidence: 99%

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Methylmalonic acid impairs cell respiration and glutamate uptake in C6 rat glioma cells

Costa

Santos

Alberto-Silva

et al. 2021

Preprint

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Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10mM) for 24 or 48 hours and cell viability, glucose consumption and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell viability after 48-hour period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-hour exposition, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposition to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Methylmalonic acid impairs cell respiration and glutamate uptake in C6 rat glioma cells

Costa

Santos

Alberto-Silva

et al. 2021

Preprint

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“…As reported in the right panel, the phosphorylation-related flux is severely affected in ROUTINE (−71.9%, p = 0.0009, n = 4) and in a less dramatic manner in OXPHOS (−18.3%, p = 0.0039, n = 4). Accordingly, the respiratory reserve (RR), consisting of the extra ATP produced by oxidative phosphorylation in case of an increased energy demand [ 41 , 42 ] was found more than halved in MPP + treated cells ( Figure 3 B, −55%, p = 0.0002, n = 4) (see also Supplementary Materials Table S3 ).…”

Section: Resultsmentioning

confidence: 99%

High-Resolution Respirometry Reveals MPP+ Mitochondrial Toxicity Mechanism in a Cellular Model of Parkinson’s Disease

Risiglione

Leggio

Cubisino

et al. 2020

IJMS

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MPP+ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson’s disease models. Within the cells, MPP+ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP+ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP+ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP+, relevant for the standardization of studies that employ this neurotoxin as a disease model.

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“…Instrumental and chemical background fluxes were calibrated as a function of the oxygen concentration using DatLab software (version 7.4.0.1, Oroboros Instruments). Rate of oxygen consumption corresponding to ROUTINE, LEAK, OXPHOS, and maximal ET capacity was corrected for the ROX and expressed as pmol/s per million cells or as FCRs relative to the maximal ET capacity [44][45][46]. Raw data were reported in Table S1.…”

Section: Analysis Of Respirometric Statesmentioning

confidence: 99%

“…The ATP-related oxygen fluxes were determined by correcting each specific state for the LEAK respiration and expressed as FCRs. The LEAK-corrected states were also used for the coupling efficiencies calculation, expressing it as a percentage of the capacity in that specific state [44,45].…”

Section: Analysis Of Respirometric Statesmentioning

confidence: 99%

Small Hexokinase 1 Peptide against Toxic SOD1 G93A Mitochondrial Accumulation in ALS Rescues the ATP-Related Respiration

et al. 2021

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Mutations in Cu/Zn Superoxide Dismutase (SOD1) gene represent one of the most common causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that specifically affects motor neurons (MNs). The dismutase-active SOD1 G93A mutant is responsible for the formation of toxic aggregates onto the mitochondrial surface, using the Voltage-Dependent Anion Channel 1 (VDAC1) as an anchor point to the organelle. VDAC1 is the master regulator of cellular bioenergetics and by binding to hexokinases (HKs) it controls apoptosis. In ALS, however, SOD1 G93A impairs VDAC1 activity and displaces HK1 from mitochondria, promoting organelle dysfunction, and cell death. Using an ALS cell model, we demonstrate that a small synthetic peptide derived from the HK1 sequence (NHK1) recovers the cell viability in a dose–response manner and the defective mitochondrial respiration profile relative to the ADP phosphorylation. This correlates with an unexpected increase of VDAC1 expression and a reduction of SOD1 mutant accumulation at the mitochondrial level. Overall, our findings provide important new insights into the development of therapeutic molecules to fight ALS and help to better define the link between altered mitochondrial metabolism and MNs death in the disease.

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High-Resolution Respirometry in Assessment of Mitochondrial Function in Neuroblastoma SH-SY5Y Intact Cells

Cited by 12 publications

References 29 publications

Methylmalonic acid impairs cell respiration and glutamate uptake in C6 rat glioma cells

Methylmalonic acid impairs cell respiration and glutamate uptake in C6 rat glioma cells

High-Resolution Respirometry Reveals MPP+ Mitochondrial Toxicity Mechanism in a Cellular Model of Parkinson’s Disease

Small Hexokinase 1 Peptide against Toxic SOD1 G93A Mitochondrial Accumulation in ALS Rescues the ATP-Related Respiration

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