Petronela Weisová

Regulation of Glucose Transporter 3 Surface Expression by the AMP-Activated Protein Kinase Mediates Tolerance to Glutamate Excitation in Neurons

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 (Δψm) 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 Δψm 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 Δψm 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.

Mitochondrial and Plasma Membrane Potential of Cultured Cerebellar Neurons during Glutamate-Induced Necrosis, Apoptosis, and Tolerance

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 ΔΨm or ΔΨp, 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 ΔΨm that was closely associated with a loss of ΔΨp 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 ΔΨm as confirmed using the ΔΨp-sensitive anionic probe DiBAC2(3). This hyperpolarization of ΔΨm was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in ΔΨm or ΔΨp at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of ΔΨp during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of ΔΨm after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in ΔΨm. Our results indicate that a hyperpolarization of ΔΨm is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress.

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

Neuronal preconditioning is a phenomenon where a previous exposure to a sub‐lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP‐activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling‐induced preconditioning in neurons, however, the molecular basis of AMPK‐mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) in a model of NMDA‐mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA‐induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti‐apoptotic BCL‐2 family protein myeloid cell leukaemia sequence 1 (MCL‐1) in AICAR‐preconditioned neurons. Interestingly, over‐expression of MCL‐1 protected neurons against NMDA‐induced excitotoxicity while MCL‐1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca2+ levels during NMDA excitation revealed that MCL‐1 over‐expressing neurons exhibited improved bioenergetics and markedly reduced Ca2+ elevations, suggesting a potential mechanism through which MCL‐1 confers neuroprotection. This study identifies MCL‐1 as a key effector of AMPK‐induced preconditioning in neurons.

Latrepirdine is a potent activator of AMP-activated protein kinase and reduces neuronal excitability

Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer’s and Huntington’s disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on TMRM uptake were largely mediated by plasma membrane hyperpolarization, precluding a purely ‘mitochondrial’ mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca2+ oscillations as well as glutamate-induced Ca2+ increases in primary neurons, and protected neurons against glutamate toxicity. In conclusion, our experiments demonstrate that latrepirdine is a potent activator of AMPK, and suggest that one of the main pharmacological activities of latrepirdine is a reduction in neuronal excitability.

TOXI-SIM-A simulation tool for the analysis of mitochondrial and plasma membrane potentials

Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe tetramethylrhodamine methyl ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Deltapsi(m)) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Deltapsi(m) and the plasma membrane (Deltapsi(p)) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Deltapsi(m) and Deltapsi(p) over time. Based on Ca(2+) responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces (http://systemsbiology.rcsi.ie/tmrm/index.html).

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.