Abstract
ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (ΔΨm; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ΔΨm was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ΔΨm. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ΔΨm with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by mitochondrial F1Fo-ATPase is insignificant.
Highlights
A variety of pathological conditions, such as ischemia, stroke, or traumatic injury, deprives neurons of oxygen and glucose supply, leading to depletion of intracellular adenosine triphosphate (ATP) levels
The time course of [ATP]c changes had a characteristic pattern exemplified in Figure 2A: the F436/F500 ratio remained at baseline level for a certain lag period, which ranged between 1 and 10 min, and dropped abruptly
We found no significant difference between the signals of ATP sensor in young and mature neuronal cultures (Figure 4), the proportion of glial cells relative to neurons increased from 16.6 ± 2.7% (5–8 DIV) to 36.6 ± 2.7% (13–14 DIV), as shown in Appendix Figure A2 (P < 0.01)
Summary
A variety of pathological conditions, such as ischemia, stroke, or traumatic injury, deprives neurons of oxygen and glucose supply, leading to depletion of intracellular adenosine triphosphate (ATP) levels (see for reviews Ames, 2000; Duchen, 2004; Iijima, 2006; Duchen and Szabadkai, 2010; Gleichmann and Mattson, 2011; Gouriou et al, 2011). Intracellular ATP is a ubiquitous “energy currency” that is crucial for energy-dependent functions in neurons including homeostasis of transmembrane ion gradients (Ames, 2000; Beal, 2000; Nicholls and Budd, 2000). It is commonly assumed that, after 1 or 2 weeks in culture, embryonic, and postnatal neurons have similar properties in terms of their response to oxidative stress, toxic doses of glutamate or glucose deprivation, i.e., to those pathology-modeling conditions that impose a heightened demand on neuronal energy suppliers (see for example, Wang and Thayer, 1996; Sattler et al, 1998; Ruiz et al, 1998; Vergun et al, 1999, 2003). No published work has reported a direct comparison of cytosolic ATP concentration ([ATP]c) levels in postnatal vs
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