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

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\u27s and Huntington\u27s 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 \u27mitochondrial\u27 mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca(2+) oscillations as well as glutamate-induced Ca(2+) 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

AMP kinase–mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis

Disturbances in cellular ion gradients by excitotoxicity promote apoptosis through activation of the Bcl-2 family member Bim

Repulsive Axon Guidance by Draxin Is Mediated by Protein Kinase B (Akt), Glycogen Synthase Kinase-3β (GSK-3β) and Microtubule-Associated Protein 1B

<div><p>Draxin is an important axon guidance cue necessary for the formation of forebrain commissures including the corpus callosum, but the molecular details of draxin signaling are unknown. To unravel how draxin signals are propagated we used murine cortical neurons and genetic and pharmacological approaches. We found that draxin-induced growth cone collapse critically depends on draxin receptors (deleted in colorectal cancer, DCC), inhibition of protein kinase B/Akt, activation of GSK-3β (glycogen synthase kinase-3β) and the presence of microtubule-associated protein MAP1B. This study, for the first time elucidates molecular events in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3β pathway essential for chemo-repulsive axon guidance cue signaling.</p></div

A model for repulsive draxin signaling.

<p>Draxin interaction with DCC triggers inactivation of the Akt pathway. This relieves GSK-3β from Akt-mediated inhibition leading to an increase in phosphorylation of MAP1B and reconfiguration of the cytoskeleton to promote growth cone collapse and inhibition of neurite extension.</p

MAP1B is required for draxin- and semaphorin 3A-induced growth cone collapse and inhibition of neurite outgrowth.

<p>A. Cortical explants from newborn wild-type or MAP1B-/- mice, cultured for 48 h in the presence or absence (control) of 10 nM draxin or 100 ng/ml semaphorin 3A (Sema3A). Scale bar = 100 μm. B. Quantification of the longest neurites of 30 explants in 3 independent experiments. C. Growth cones of cortical neurons from newborn wild-type or MAP1B-/- mice cultured for 60 h, treated for 1 h with 100 nM draxin or 30 min with 100 ng/ml semaphorin 3A (Sema3A), fixed and stained for F-actin. Scale bar = 10 μm. D. Percentage of collapsed growth cones. For each experimental condition the growth cones of 30 neurons in each of 3 independent experiments were evaluated.</p

Draxin-induced growth cone collapse is mediated through DCC.

<p>A. Growth cones of cortical neurons from newborn mice cultured for 60 h, pretreated for 1 h with function blocking anti-DCC antibodies (anti-DCC) or unrelated IgGs at the indicated concentrations followed by addition of draxin for 1 h as indicated, fixation and staining for F-actin. Scale bar = 10 μm. B. Quantification of growth cone collapse. For each experimental condition the growth cones of 40 neurons in each of 3 independent experiments were evaluated.</p

Draxin-induced growth cone collapse is dependent on GSK-3β activity and mimicked by inhibition of PI3K.

<p>A. Growth cones of cortical neurons from newborn wild-type mice cultured for 60 h, pre-treated with solvent (DMSO) or the GSK-3β inhibitor SB216763 or the PI3K inhibitor wortmannin for 1 h followed by addition of draxin for 1 h as indicated. Cells were fixed and stained for F-actin. Scale bar = 10 μm. B and C. Quantification of growth cone collapse. For each experimental condition the growth cones of 40 neurons in each of 3 independent experiments were evaluated.</p

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.</p