Mitochondria‐coupled glucose phosphorylation develops after birth to modulate H₂O₂ release and calcium handling in rat brain

Abstract

The adult brain is a high-glucose and oxygen-dependent organ, with an extremely organized network of cells and large energyconsuming synapses. To reach this level of organization, early stages in development must include an efficient control of cellular events and regulation of intracellular signaling molecules and ions such as hydrogen peroxide (H 2 O 2 ) and calcium (Ca 2+ ), but in cerebral tissue, these mechanisms of regulation are still poorly understood. Hexokinase (HK) is the first enzyme in the metabolism of glucose and, when bound to mitochondria (mtHK), it has been proposed to have a role in modulation of mitochondrial H 2 O 2 generation and Ca 2+ handling. Here, we have investigated how mtHK modulates these signals in the mitochondrial context during postnatal development of the mouse brain. Using high-resolution respirometry, western blot analysis, spectrometry and resorufin, and Calcium Green fluorescence assays with brain mitochondria purified postnatally from day 1 to day 60, we demonstrate that brain HK increases its coupling to mitochondria and to oxidative phosphorylation to induce a cycle of ADP entry/ATP exit of the mitochondrial matrix that leads to efficient control over H 2 O 2 generation and Ca 2+ uptake during development until reaching plateau at day 21. This contrasts sharply with the antioxidant enzymes, which do not increase as mitochondrial H 2 O 2 generation escalates. These results suggest that, as its use of glucose increases, the brain couples HK to mitochondria to improve glucose metabolism, redox balance and Ca 2+ signaling during development, positioning mitochondria-bound hexokinase as a hub for intracellular signaling control. Abbreviations used: b-HB, b-hydroxybutyrate; b-HBM, b-hydroxybutyrate plus malate; 2-DG, 2-Deoxyglucose; Ap5A, P1,P5-Di(adenosine-5 0 ) pentaphosphate; DTNB, 5,5 0 -Dithiobis(2-nitrobenzoic acid); FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; G6PDH, glucose-6phosphate dehydrogenase; mΔw, mitochondrial membrane potential; mETS, mitochondrial electron transport system; mPTP, mitochondrial permeability transition pore; mtHK, mitochondrial bound hexokinase; ODC, oxoglutarate dehydrogenase complex; Omy, oligomicyn; PDC, pyruvate dehydrogenase complex; ROS, reactive oxygen species; RRID, research resource identifier. 624

MicroRNAs in the Onset of Schizophrenia

“…Markers of oxidative stress increase at the onset of adolescence and during adult aging, both in the brain and periphery [124][125][126]. In brain-derived mitochondria, the rate of aerobic respiration increases during early postnatal and adolescent development, reaching adult levels at ~P60 (end of adolescence/onset of adulthood) in rats [127]. Oxidative stress occurs when the reactive oxygen species formed as natural byproducts of aerobic respiration accumulate to deleterious levels and cause damage to DNA, lipids, and other cellular components susceptible to oxidation.…”

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC‐Derived Astrocytes

“…During neurodevelopment and in adult brain cells, ROS have important signaling roles in proliferation, differentiation, and migration (Kennedy, Sandiford, Skerjanc, & Li, 2012;Wilson, Muñoz-Palma, & González-Billault, 2018). In neural cells, most hexokinase is bound to OMM, possibly due to the modulation of ROS generation associated with the activity of mt-HK (da-Silva et al, 2004;de-Souza-Ferreira, Rios-Neto, Martins, & Galina, 2019). Therefore, measuring mt-HK enzymatic activity, particularly in human neural cells, constitutes an interesting strategy to assess mitochondrial function more fully.…”

“…Despite the complexity of mitochondrial calcium dynamics modulation, there are some possible explanations for variations in the functional parameters. It has been recently shown that activation of mitochondrial hexokinase, an enzyme bound to the outer mitochondrial membrane that phosphorylates glucose in a reaction coupled to OXPHOS, increases the uptake of calcium by brain mitochondria (de-Souza-Ferreira et al, 2019), probably due to the modulation of ADP/ATP cycling and of physiological mPTP opening (Jonas et al, 1999). Therefore, if an improvement in the uptake of calcium influx rate is observed in astrocytes or other neural cells in the presence of glucose, it may reveal the stimulation of mt-HK activity or another system that modulates nucleotide cycling.…”

“…Pharmacological drugs or neurological conditions that can be recapitulated with iPSC-derived neural cells may affect the activity of mt-HK, which may reflect alterations in cellular glucose metabolism. Since mt-HK is an important modulator of ROS generation in brain cells (da-Silva et al, 2004;de-Souza-Ferreira et al, 2019), associating the measurement of mt-HK activity with evaluation of ROS levels is an interesting approach. In a condition where mt-HK activity of neural cells is stimulated, probably the generation of ROS by mitochondria is lower.…”

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells

“…Therefore, mitochondrial ROS and their regulation by mt-HK are important for neurodevelopment [ 28 ]. Our group showed that mt-HK activity linearly increases throughout neurodevelopmental stages [ 29 ], correlating with oxidative metabolism and mitochondrial ROS production, unlike classical antioxidant systems [ 30 ]. This observation suggests that mt-HK may also modulate mitochondrial ROS during brain development.…”