Regulation of neuronal bioenergy homeostasis by glutamate

Abstract

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.

Figure 1

Application of glutamate causes a reduction in ATP abundance in cultured cortical neurons. (A) A standard curve of ATP assay showing linear relationship between ATP levels and Luminometer measurement values. (B) Time course of glutamate treatment. 10 min glutamate incubation induced a significant reduction in ATP levels, which was further reduced by 60 min treatment. (C) Glutamate dose response. Significant changes in ATP amount could be induced by as low as 10 μM glutamate, and a plateau was reached by 50 μM glutamate. *P<0.05, student’s t test.

Figure 2

Glutamate effect in ATP abundance is not caused by elevated neuronal firing. (A) Blockade of energy synthesis process by KCN lead to a dramatic reduction in ATP levels. In the presence of KCN, glutamate treatment remained to be able to cause ATP reduction. (B) Changes in ATP abundance following KCl-induced neuronal excitation were blocked by TTX; in contrast, blockade of neuronal firing had no effect on glutamate-dependent ATP regulation. *P<0.05, student’s t test.

Figure 3

Glutamate effect in ATP regulation is mediated mainly by NMDARs. (A) Neurons were incubated with glutamate in the presence of antagonists against AMPAR, NMDAR and mGluR, applied individually for one type of receptor, or together as a mixed antagonist cocktail (AC) to block all receptors. AC and NMDAR antagonist APV, but not AMPAR antagonist CNQX or mGluR antagonist MCPG, abolished the glutamate effect, indicating a key role for NMDARs (n=4). (B) Neurons were treated with glutamate or an agonist specific for AMPAR (AMPA), NMDAR (NMDA) and mGluR (DHPG), respectively. NMDA induced an ATP reduction to a level similar to that of glutamate treatment, whereas AMPA and DHPG had no effect (n=3). (C) Glutamate and receptor antagonists had no effect on ATP levels in HEK cells (n=3). *P<0.05, student’s t test.

Figure 4

Involvement of signaling pathways in glutamate effects. Neurons were treated with glutamate in the presence of varied drugs to inhibitor the activity of PKA, PI3K, CaMKII and tyrosine kinases. None of the inhibitors showed effect on the glutamate-induced ATP reduction. *P<0.05, student’s t test.

Figure 5

Effect of synaptic glutamate accumulation on ATP abundance. Incubation of cultured neurons with glutamate transporter inhibitor TBOA caused a marked reduction in ATP levels, which was blocked by receptor antagonist cocktail (AC). Co-application of TBOA and glutamate reduced ATP to a level similar to that by either glutamate or TBOA alone, suggesting a saturation by a single treatment, or a shared cellular process triggered by global and synaptic glutamate stimulation. *P<0.05, student’s t test.

Figure 6

The role of sodium pumps in glutamate regulation of ATP abundance. Blocking of sodium pump by ouabain (Oua) caused a marked increase in ATP amount. In the presence of ouabain, glutamate failed to induce a reduction in ATP amount. *P<0.05, student’s t test.

Figure 7

Glutamate effect on ATP does not result from gap junction-mediated ATP release. In the presence of a gap junction blocker carbenoxolene (Carb), glutamate incubation reduced ATP to a level comparable to that by glutamate alone. *P<0.05, student’s t test.

Figure 8

Effect of glutamate on glial ATP. (A) Regular neuronal cultures (containing both neurons and glia) and glial cultures (neuron free) were lysed and analyzed by western blotting. Regular mixed neuronal cultures contained high levels of AMPAR subunit GluA1, but only a low level of the glial marker protein GFAP. In contrast, the glial cultures were enriched in GFAP, but only expressed minimal levels of glutamate receptors. (B) In glial cultures, glutamate induced a modest, but significant reduction in ATP amount. The change was blocked by NMDAR antagonist APV, but not AMPAR antagonist CNQX. *P<0.05, student’s t test.