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

Weisová P, Alvarez SP, Kilbride SM, Anilkumar U, Baumann B, Jordán J, Bernas T, Huber HJ, Düssmann H, Prehn JH - Transl Psychiatry (2013)

Bottom Line: Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology and Medical Physics, Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin, Ireland [2] Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

ABSTRACT
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 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.

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Related in: MedlinePlus

Latrepirdine pretretment attenuates the increase in cytosolic Ca2+ during glutamate excitation and reduces spontaneous Ca2+ oscillations. (a) Average single-cell traces of changes in fluorescence intensity of the cytosolic Ca2+ indicator Fluo-4 AM in response to glutamate excitation. CGNs pretreated with latrepirdine (0.1 nM for 24 h were loaded with Fluo-4 AM (3 μM) and mounted on the confocal microscope stage. Glutamate excitation (glutamate/glycine [100 μM/10 μM] for 10 min immediately followed by addition of MK 801 [2.5 μM]) was induced as indicated. Analysis was carried out using Metamorph software and average pixel intensity per population at each timepoint is shown. (b) Quantification of area under the Fluo-4 AM curve during glutamate excitation in prolonged latrepirdine-pretreated neurons. Vehicle: n=30 cells; latrepirdine: (n=45 cells). Data are shown as mean±s.e.m. *P<0.001 compared with vehicle-pretreated neurons that were glutamate treated. (c) Murine cortical neurons were cultivated on glass bottom dishes, incubated with 5 μM Fluo-4-AM for 45 min at 37 °C, washed, perfused with experimental buffer supplemented with 2 mM MgCl2 and placed on the heated stage of a LSM 5live microscope. Images were taken at 5 Hz, optical slice thickness set to 3.5 μm. The buffer was replaced with MgCl2-free buffer at time 0 and either vehicle or latrepirdine (0.1 nM) was added after 120 s of imaging as indicated. Typical Fluo-4 kinetics are shown as change in fluorescence intensity divided by the mean overall fluorescence intensity (ΔF/F). (d) Quantification of Ca2+ spike frequency after MgCl2 washout, treatment with latrepirdine (0.1 nM, n=176 cells) or (e) AICAR (0.1 mM) (n=88 cells) addition followed by complete block using tetrodotoxin (TTX, 1 μM) (significant difference P<0.05, paired t-test). (f) Quantification of changes of the Ca2+-spiking activity due to addition of latrepirdine, AICAR, or vehicle (Control, n=134 cells, latrepirdine, n=176 cells, AICAR n=88 cells, Kruskal–Wallis and subsequent Mann–Whitney tests showed a significant difference in latrepirdine and AICAR compared with control (*) but no significant difference in latrepirdine compared with AICAR (ns).
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fig5: Latrepirdine pretretment attenuates the increase in cytosolic Ca2+ during glutamate excitation and reduces spontaneous Ca2+ oscillations. (a) Average single-cell traces of changes in fluorescence intensity of the cytosolic Ca2+ indicator Fluo-4 AM in response to glutamate excitation. CGNs pretreated with latrepirdine (0.1 nM for 24 h were loaded with Fluo-4 AM (3 μM) and mounted on the confocal microscope stage. Glutamate excitation (glutamate/glycine [100 μM/10 μM] for 10 min immediately followed by addition of MK 801 [2.5 μM]) was induced as indicated. Analysis was carried out using Metamorph software and average pixel intensity per population at each timepoint is shown. (b) Quantification of area under the Fluo-4 AM curve during glutamate excitation in prolonged latrepirdine-pretreated neurons. Vehicle: n=30 cells; latrepirdine: (n=45 cells). Data are shown as mean±s.e.m. *P<0.001 compared with vehicle-pretreated neurons that were glutamate treated. (c) Murine cortical neurons were cultivated on glass bottom dishes, incubated with 5 μM Fluo-4-AM for 45 min at 37 °C, washed, perfused with experimental buffer supplemented with 2 mM MgCl2 and placed on the heated stage of a LSM 5live microscope. Images were taken at 5 Hz, optical slice thickness set to 3.5 μm. The buffer was replaced with MgCl2-free buffer at time 0 and either vehicle or latrepirdine (0.1 nM) was added after 120 s of imaging as indicated. Typical Fluo-4 kinetics are shown as change in fluorescence intensity divided by the mean overall fluorescence intensity (ΔF/F). (d) Quantification of Ca2+ spike frequency after MgCl2 washout, treatment with latrepirdine (0.1 nM, n=176 cells) or (e) AICAR (0.1 mM) (n=88 cells) addition followed by complete block using tetrodotoxin (TTX, 1 μM) (significant difference P<0.05, paired t-test). (f) Quantification of changes of the Ca2+-spiking activity due to addition of latrepirdine, AICAR, or vehicle (Control, n=134 cells, latrepirdine, n=176 cells, AICAR n=88 cells, Kruskal–Wallis and subsequent Mann–Whitney tests showed a significant difference in latrepirdine and AICAR compared with control (*) but no significant difference in latrepirdine compared with AICAR (ns).

Mentions: Glutamate excitotoxicity is characterized by excessive Ca2+ influx through NMDA receptors, leading to intracellular Ca2+ overload.35 Indeed, glutamate-induced Ca2+ elevations critically depend both on the magnitude of plasma membrane potential depolarization,36 as well as ATP-dependent Ca2+ extrusion.37 Our observations of plasma membrane hyperpolarization and the changes in cellular bioenergetics in response to latrepirdine posed the question whether protection by pretreatment with latrepirdine may be mediated by reduced neuronal Ca2+ overloading during glutamate excitation. CGN neurons were pretreated with latrepirdine (0.1 nM), and changes in cytosolic Ca2+ levels were monitored by confocal microscopy using Fluo-4 A.M. CGNs pretreated for 24 h with latrepirdine, and then exposed to glutamate and glycine (100 μM/10 μM for 10 min) significantly attenuated cytosolic Ca2+ influx (Figures 5a and b). Quantification of peak fluo-4 fluorescence (Figure 5b) during the glutamate exposure showed a robust attenuation of Ca2+ influx in CGN neurons pretreated with latrepirdine (0.1 nM) for 24 h compared with vehicle-pretreated neurons. This finding was furthermore confirmed by the observation that pharmacological activation of AMPK with AICAR (0.1 mM, 24 h before glutamate excitation) also led to a significant attenuation of cytosolic Ca2+ levels during NMDA receptor overactivation in cortical neurons (NMDA alone: 5516.72 ± 1126.52 fl. int. units, n=70 cells vs. AICAR pretreated 3174.34±1152.78 fluorescence intensity units, n=67 cells, P<0.001). Collectively, these data suggested that pharmacological AMPK activation with latrepirdine pretreatment affects Ca2+ handling in primary neurons in response to glutamate excitotoxicity. Interestingly, acute pretreatment with latrepirdine (0.1 nM, 10 min before glutamate) did not attenuate Ca2+ influx (Supplementary Figures 4A and B), suggesting that latrepirdine did not act directly on glutamate receptors.


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

Weisová P, Alvarez SP, Kilbride SM, Anilkumar U, Baumann B, Jordán J, Bernas T, Huber HJ, Düssmann H, Prehn JH - Transl Psychiatry (2013)

Latrepirdine pretretment attenuates the increase in cytosolic Ca2+ during glutamate excitation and reduces spontaneous Ca2+ oscillations. (a) Average single-cell traces of changes in fluorescence intensity of the cytosolic Ca2+ indicator Fluo-4 AM in response to glutamate excitation. CGNs pretreated with latrepirdine (0.1 nM for 24 h were loaded with Fluo-4 AM (3 μM) and mounted on the confocal microscope stage. Glutamate excitation (glutamate/glycine [100 μM/10 μM] for 10 min immediately followed by addition of MK 801 [2.5 μM]) was induced as indicated. Analysis was carried out using Metamorph software and average pixel intensity per population at each timepoint is shown. (b) Quantification of area under the Fluo-4 AM curve during glutamate excitation in prolonged latrepirdine-pretreated neurons. Vehicle: n=30 cells; latrepirdine: (n=45 cells). Data are shown as mean±s.e.m. *P<0.001 compared with vehicle-pretreated neurons that were glutamate treated. (c) Murine cortical neurons were cultivated on glass bottom dishes, incubated with 5 μM Fluo-4-AM for 45 min at 37 °C, washed, perfused with experimental buffer supplemented with 2 mM MgCl2 and placed on the heated stage of a LSM 5live microscope. Images were taken at 5 Hz, optical slice thickness set to 3.5 μm. The buffer was replaced with MgCl2-free buffer at time 0 and either vehicle or latrepirdine (0.1 nM) was added after 120 s of imaging as indicated. Typical Fluo-4 kinetics are shown as change in fluorescence intensity divided by the mean overall fluorescence intensity (ΔF/F). (d) Quantification of Ca2+ spike frequency after MgCl2 washout, treatment with latrepirdine (0.1 nM, n=176 cells) or (e) AICAR (0.1 mM) (n=88 cells) addition followed by complete block using tetrodotoxin (TTX, 1 μM) (significant difference P<0.05, paired t-test). (f) Quantification of changes of the Ca2+-spiking activity due to addition of latrepirdine, AICAR, or vehicle (Control, n=134 cells, latrepirdine, n=176 cells, AICAR n=88 cells, Kruskal–Wallis and subsequent Mann–Whitney tests showed a significant difference in latrepirdine and AICAR compared with control (*) but no significant difference in latrepirdine compared with AICAR (ns).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig5: Latrepirdine pretretment attenuates the increase in cytosolic Ca2+ during glutamate excitation and reduces spontaneous Ca2+ oscillations. (a) Average single-cell traces of changes in fluorescence intensity of the cytosolic Ca2+ indicator Fluo-4 AM in response to glutamate excitation. CGNs pretreated with latrepirdine (0.1 nM for 24 h were loaded with Fluo-4 AM (3 μM) and mounted on the confocal microscope stage. Glutamate excitation (glutamate/glycine [100 μM/10 μM] for 10 min immediately followed by addition of MK 801 [2.5 μM]) was induced as indicated. Analysis was carried out using Metamorph software and average pixel intensity per population at each timepoint is shown. (b) Quantification of area under the Fluo-4 AM curve during glutamate excitation in prolonged latrepirdine-pretreated neurons. Vehicle: n=30 cells; latrepirdine: (n=45 cells). Data are shown as mean±s.e.m. *P<0.001 compared with vehicle-pretreated neurons that were glutamate treated. (c) Murine cortical neurons were cultivated on glass bottom dishes, incubated with 5 μM Fluo-4-AM for 45 min at 37 °C, washed, perfused with experimental buffer supplemented with 2 mM MgCl2 and placed on the heated stage of a LSM 5live microscope. Images were taken at 5 Hz, optical slice thickness set to 3.5 μm. The buffer was replaced with MgCl2-free buffer at time 0 and either vehicle or latrepirdine (0.1 nM) was added after 120 s of imaging as indicated. Typical Fluo-4 kinetics are shown as change in fluorescence intensity divided by the mean overall fluorescence intensity (ΔF/F). (d) Quantification of Ca2+ spike frequency after MgCl2 washout, treatment with latrepirdine (0.1 nM, n=176 cells) or (e) AICAR (0.1 mM) (n=88 cells) addition followed by complete block using tetrodotoxin (TTX, 1 μM) (significant difference P<0.05, paired t-test). (f) Quantification of changes of the Ca2+-spiking activity due to addition of latrepirdine, AICAR, or vehicle (Control, n=134 cells, latrepirdine, n=176 cells, AICAR n=88 cells, Kruskal–Wallis and subsequent Mann–Whitney tests showed a significant difference in latrepirdine and AICAR compared with control (*) but no significant difference in latrepirdine compared with AICAR (ns).
Mentions: Glutamate excitotoxicity is characterized by excessive Ca2+ influx through NMDA receptors, leading to intracellular Ca2+ overload.35 Indeed, glutamate-induced Ca2+ elevations critically depend both on the magnitude of plasma membrane potential depolarization,36 as well as ATP-dependent Ca2+ extrusion.37 Our observations of plasma membrane hyperpolarization and the changes in cellular bioenergetics in response to latrepirdine posed the question whether protection by pretreatment with latrepirdine may be mediated by reduced neuronal Ca2+ overloading during glutamate excitation. CGN neurons were pretreated with latrepirdine (0.1 nM), and changes in cytosolic Ca2+ levels were monitored by confocal microscopy using Fluo-4 A.M. CGNs pretreated for 24 h with latrepirdine, and then exposed to glutamate and glycine (100 μM/10 μM for 10 min) significantly attenuated cytosolic Ca2+ influx (Figures 5a and b). Quantification of peak fluo-4 fluorescence (Figure 5b) during the glutamate exposure showed a robust attenuation of Ca2+ influx in CGN neurons pretreated with latrepirdine (0.1 nM) for 24 h compared with vehicle-pretreated neurons. This finding was furthermore confirmed by the observation that pharmacological activation of AMPK with AICAR (0.1 mM, 24 h before glutamate excitation) also led to a significant attenuation of cytosolic Ca2+ levels during NMDA receptor overactivation in cortical neurons (NMDA alone: 5516.72 ± 1126.52 fl. int. units, n=70 cells vs. AICAR pretreated 3174.34±1152.78 fluorescence intensity units, n=67 cells, P<0.001). Collectively, these data suggested that pharmacological AMPK activation with latrepirdine pretreatment affects Ca2+ handling in primary neurons in response to glutamate excitotoxicity. Interestingly, acute pretreatment with latrepirdine (0.1 nM, 10 min before glutamate) did not attenuate Ca2+ influx (Supplementary Figures 4A and B), suggesting that latrepirdine did not act directly on glutamate receptors.

Bottom Line: Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology and Medical Physics, Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin, Ireland [2] Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

ABSTRACT
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 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.

Show MeSH
Related in: MedlinePlus