No abstract

The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Ischemic and excitotoxic events within the brain result in rapid and often unfavorable depletions in neuronal energy levels. Here, we investigated the signaling pathways activated in response to the energetic stress created by transient glutamate excitation in cerebellar granule neurons. We characterized a glucose dependent hyperpolarization of the mitochondrial membrane potential (⌬ m ) in the majority of neurons after transient glutamate excitation. Expression levels of the primary neuronal glucose transporters (GLUTs) isoforms 1, 3, 4, and 8 were found to be unaltered within a 24 h period after excitation. However, a significant increase only in GLUT3 surface expression was identified 30 min after excitation, with this high surface expression remaining significantly above control levels in many neurons for up to 4 h. Glutamate excitation induced a rapid alteration in the AMP:ATP ratio that was associated with the activation of the AMP-activated protein kinase (AMPK). Interestingly, pharmacological activation of AMPK with AICAR (5-aminoimidazole-4-carboxamide riboside) alone also increased GLUT3 surface expression, with a hyperpolarization of ⌬ m evident in many neurons. Notably, inhibition of the CaMKK (calmodulin-dependent protein kinase kinase) had little affect on GLUT translocation, whereas the inhibition or knockdown of AMPK (compound C, siRNA) activity prevented GLUT3 translocation to the cell surface after glutamate excitation. Furthermore, gene silencing of GLUT3 eradicated the increase in ⌬ m associated with transient glutamate excitation and potently sensitized neurons to excitotoxicity. In summary, our data suggest that the activation of AMPK and its regulation of cell surface GLUT3 expression is critical in mediating neuronal tolerance to excitotoxicity.

Mitochondria within cultured rat cerebellar granule cells have a complex influence on cytoplasmic free Ca2+ ([Ca2+]c) responses to glutamate. A decreased initial [Ca2+]c elevation in cells whose mitochondria are depolarized by inhibition of the ATP synthase and respiratory chain (conditions which avoid ATP depletion) was attributed to enhanced Ca2+ extrusion from the cell rather than inhibited Ca2+ entry via the NMDA receptor. Even in the presence of elevated extracellular Ca2+, when [Ca2+]c responses were restored to control values, such cells showed resistance to acute excitotoxicity, defined as a delayed cytoplasmic Ca2+ deregulation (DCD) during glutamate exposure. DCD was a function of the duration of mitochondrial polarization in the presence of glutamate rather than the total period of glutamate exposure. Once initiated, DCD could not be reversed by NMDA receptor inhibition. In the absence of ATP synthase inhibition, respiratory chain inhibitors produced an immediate Ca2+ deregulation (ICD), ascribed to an ATP deficit. In contrast to DCD, ICD could be reversed by subsequent ATP synthase inhibition with or without additional NMDA receptor blockade. DCD could not be ascribed to the failure of an ATP yielding metabolic pathway. It is concluded that mitochondria can control Ca2+ extrusion from glutamate-exposed granule cells by the plasma membrane in three ways: by competing with efflux pathways for Ca2+, by restricting ATP supply, and by inducing a delayed failure of Ca2+ extrusion. Inhibitors of the mitochondrial permeability transition only marginally delayed the onset of DCD.

A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either ⌬⌿ m or ⌬⌿ p , thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of ⌬⌿ m that was closely associated with a loss of ⌬⌿ p and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of ⌬⌿ m as confirmed using the ⌬⌿ p -sensitive anionic probe DiBAC 2 (3). This hyperpolarization of ⌬⌿ m was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in ⌬⌿ m or ⌬⌿ p at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of ⌬⌿ p during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of ⌬⌿ m after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in ⌬⌿ m . Our results indicate that a hyperpolarization of ⌬⌿ m is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.