Supplementary Information

Materials and Methods

Materials

Fetal calf serum and minimal essential medium were from Invitrogen (Bio Sciences, Dun Laoghaire, Ireland). Glutamate and glycine were from Sigma Aldrich (Tallaght, Dublin, Ireland). Fluo-4 acetomethyl ester (AM), tetramethylrhodamine methyl ester (TMRM), [Bis-(1,3-diethylthiobarbituric acid)trimethine oxonol] (DisBAC(2)3), MitoTracker Green-FMTM, propidium iodide (PI) and Hoechst 33258 were purchased from Invitrogen. Compound C was obtained from Calbiochem (Merck Biosciences, Nottingham, UK). Aminoimidazole carboxamide ribonucleotide (AICAR) was from Cell Signaling (Isis, Wicklow, Ireland) and Mk-801 from Fluka (Dublin, Ireland). Latrepirdine (Dimebon) was provided by Medivation Inc. (San Francisco, CA, U.S.A.).

Monitoring of changes in mitochondrial (Δψm), plasma (Δψp) membrane potential and cytosolic Ca2+ levels and Ca2+ oscillations by time-lapse confocal microscopy.

Primary cerebellar granule neurons cultured on Willco dishes were coloaded with Fluo-4 AM (3 μm) for detection of changes in cytosolic Ca2+, and TMRM (10 nM) for detection of mitochondrial TMRM uptake, an indicator of changes in Δψm., but also subject to changes in Δψp. Cells were loaded with the dyes at 37°C in the dark for 30 min in experimental buffer containing (in mm): 120 NaCl, 3.5 KCl, 0.4 KH2PO4, 20 HEPES, 5 NaHCO3, 1.2 Na2SO4, 1.2 CaCl2, and 15 glucose, pH 7.4. The cells were washed and bathed in 2 ml of experimental buffer containing TMRM, and a thin layer of embryo tested mineral oil (Sigma, Ireland) was added to prevent evaporation. The Willco dishes with neurons were mounted on the stage of an LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 63×, 1.4 NA oil-immersion objective and a thermostatically regulated chamber (Carl Zeiss, Jena, Germany). Neurons were incubated either with latrepirdine (0.1 nM in distilled water) or vehicle for 10 min for measurement of ‘acute’ responses, or for 24 h prior to glutamate exposure (long-term treatment). After 30 min equilibration time, neurons were exposed to glutamate and glycine (100 and 10 µM) for 10 min, with MK-801(10 µM) added to inhibit NMDA receptor activation. Fluo-4 AM (3 µM) was excited at 488 nm, and the emission was collected through a 505–550 nm barrier filter; TMRM was excited at 543 nm, and the emission was collected through a 560 nm long-pass filter. In another set of experiments, the fluorescent probe DisBAC2(3) (1µM) was employed to characterize exclusively the changes in Δψp 1. CGNs, transfected with expressions vectors shRNA scrambled GFP, shRNA-AMPK α1/α2 GFP, shRNA LKB1 GFP, or shRNA CaMKKβ GFP and loaded with TMRM (10 nm) and DisBAC2(3) (1 µM) in experimental buffer, were placed on the stage of an LSM 710 confocal microscope equipped with a 63×, 1.3 NA oil-immersion objective and a thermostatically regulated chamber set at 37°C (Zeiss). After a 30 min equilibration time, Dimebon was added to the experimental buffer. TMRM was excited at 561 nm, and the emission was collected by a 575 nm long-pass filter. DisBAC2(3) was excited at 488 nm with an argon laser (1%), and the emission was collected through a 530–600 nm barrier filter. Images were captured every 1 min throughout a 240 min experiment. Experiments were terminated by addition of FCCP (10 µM) or ionomycin (10 µM). Control experiments under these conditions were also performed to ensure that phototoxicity had a negligible impact. All images were processed using MetaMorph Software version 7.5 (Universal Imaging), and the data were presented normalised to baseline. For cytosolic Ca2+ oscillations, an LSM 5live microscope equipped with a 40x, 1.3NA oil-immersion objective (Carl Zeiss) was used. Murine cortical neurons at DIV 9 were loaded with Fluo4AM (5 mM) for 45 min at 37°C, washed, and transferred into experimental buffer. Neuron cultures were then transferred to a heated stage insert on an LSM5live confocal microscope, and single-cell Ca2+ imaging was performed with a frequency of 5Hz in the presence and absence of Mg2+ (2 mM).

Analysis of mitochondrial (Δψm) and plasma (Δψp) membrane potential kinetics

Membrane potential kinetics were analyzed according to Ward et al. (2007) 2. Briefly, the Nernstian distribution of DisBAC2(3) was determined to calculate changes in Δψp, which was subsequently used to calculate the Nernstian distribution of TMRM. For a given time interval (Dt), we assumed that the dyes were in equilibrium, and that changes in TMRM and DisBAC2(3) concentrations was reflecting the current membrane potential. Therefore F(DisBAC2(3),t) = F(DisBAC2(3),t-Dt) exp (DYp/RTF) allows us to calculate the Δψp kinetics from the DisBAC2(3) traces (with F(): Fluorescence intensity, t: time, R: Rydberg’s constant, T: Temperature, F: Faraday’s constant).

Δψp kinetics were subsequently used to calculate the Δψp-sensitive proportion of the TMRM traces with the knowledge of the mitochondrial volume fraction (frac) as follows: F(TMRM, tDt) = qVcytCcyt + VmitCmit = qVcyt (Ccyt + fracCmit) = qVcyt(1+frac exp(Yp/RTF)

(with q: brightness of the fluorescent dye, V: Volume, C: Concentration and the indices cyt: cytosol, mit: mitochondrial).

The TMRM fluorescence intensity for the subsequent time was calculated accordingly: F(TMRM,t) = qVcyt(1+frac exp((Yp+DYp)/RTF)) exp((-Ym+DYm)/RTF). In order to calculate changes in F(TMRM) induced by Δψp changes alone, we set DYm=0 which lead us to F(TMRM, t)/F(TMRM, tDt) =(1+frac exp((Yp+DYp)/RTF))/ (1+frac exp((Yp)/RTF)). This equation was used to calculate the TMRM fluorescence intensity shown in Figure 3D.

SDS-PAGE and Immunoblotting.

Preparation of neuronal cell lysates and immunoblotting was performed as following. The Pierce BCA Micro Protein assay kit was used to determine the protein concentration. Samples were prepared with 2-mercaptoethanol and denatured by heating at 95 °C for 10 min. The proteins were separated on 10% polyacrylamide gels for pAMPK (Thr 172), AMPK, LKB1, and CaMKKβ; 6% gels for pACC (Ser 79) and ACC or 15% gels for COX IV by SDS-PAGE and were transferred to nitrocellulose membranes (Protean BA 85; Schleicher & Schuell, Dassel, Germany). Membranes were blocked with 5 % non-fat milk for 60 min at room temperature. The blots were probed with the following antibodies: mouse monoclonal COX IV (Invitrogen A21348, 1:1000), rabbit polyclonal phospho-AMPK (Thr 172), AMPKα, LKB1 and CaMKKβ antibodies (Cell Signaling Technology, 1:1000); and a mouse, monoclonal anti-actin antibody (Sigma, Tallaght, Dublin, Ireland) overnight at 4°C. Membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times for 10 min. Horseradish peroxidase-conjugated secondary antibodies diluted 1:10,000 (Pierce) were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and imaged using a FujiFilm LAS-3000 imaging system (FujiFilm).

Quantification of Mitochondrial Mass

Amount of mitochondria in neurons was quantified using MitoTracker Green FM (Molecular Probes, Eugene, OR) 3. CGNs were loaded in cultured media with 150 nM MitoTracker Green FM for 30 min at 37° C. After incubation the medium was aspirated, neurons were trypsinised and centrifuged at 6,000 rpm for 3 min. After resuspending in PBS, the forward scatter versus side scatter area was analysed for population of live neurons by flow cytometry. A minimum of 104 events per each sample were acquired with flow cytometric analysis performed on the CyFlow ML (Münster, Germany) and analysed by FloMax software.

Statistics.

Data are given as means ± SEM. For statistical comparison, one-way analysis of variance between groups and Student-Newman-Keuls post hoc test were carried out on SPSS software (SPSS GmbH Software, Munich, Germany). Where the p value was < 0.05, groups were considered to be significantly different.

Supplementary Figure 1: Latrepirdine is not protective when applied acutely (10 min 0.1 nM) against glutamate excitoxicity

Murine cerebellar granular neurons were plated in 24 well plates and cells were exposed to glutamate/glycine 100 µM/10 µM for 10 min in the presence of Latrepirdine as indicated. After treatment, cells were washed twice with high Mg2+ buffer and incubated in preconditioned medium for a further 24 h. More than one thousand cells were imaged in each well and the pyknotic nuclei were counted as apoptotic, as determined by Hoechst 33358 staining (1 µg/mL) and expressed as a percentage of total. Data are presented as mean ± SEM carried out in triplicate in three separate wells. Experiments were performed twice from separate preparations with similar results obtained. ns indicates no significant difference between indicated groups

Supplementary Figure 2: Effects of Latrepirdine pre-treatment (0.1 nM for 24 h) on Δψm during glutamate excitotoxicity

Primary neurons pretreated with latrepirdine (0.1 nM for 24 h or for 10 min) as described above were loaded with TMRM (10 nM) in experimental buffer and kept for 30 min at 37 °C in dark. Subsequently neurons were mounted on a stage of Zeiss LSM 510 confocal microscope and glutamate excitotoxicity (100 µM / 10 µM for 10 min followed by addition of MK 801) was induced and monitored in real time. Quantification of TMRM fluorescence prior glutamate injury (in baseline) and during excitotoxicity in vehicle (n = 54) or Dimebon (0.1 nM for 24 h) pretreated neurons (n = 61). Data are presented as mean ± SEM. *p < 0.01 difference between glutamate only treated and latrepirdine (0.01) pretreated glutamate pretreated neurons in baseline. Ns – no significant difference in Δψm levels between vehicle vs latrepirdine treated neurons during glutamate excitotoxicity. Experiment was repeated from three independent cultures with similar data obtained.

Supplementary Figure 3: Latrepirdine increases TMRM fluorescence intensity without affecting mitochondrial biogenesis

A) CGNs 24 h after treatment with latrepirdine (0.1 nM) were lysed and examined for COX IV expression levels by Western Blotting. Actin was used as a loading control. This experiment was repeated twice with similar results. B) Latrepirdine treated neurons (0.1 nM for 24 h) were loaded with MitoTracker Green FM (100 nM for 45 min) in media, and fluorescence intensity, indicative of mitochondrial mass, was quantified by flow cytometry. The mean fluorescence intensity of each population was obtained, and data shown represent mean ± SEM of n = 3 populations per treatment. No significant differences between groups change was detected. C) After treatment of CGNs with Latrepirdine (0.1 nM for 24 h), the mRNA expression of pgc-1α and tfam were examined by real-time qPCR. Data are shown as mean ± SEM from three independent cultures (n = 3 experiments in triplicate). ns indicates that no significant difference was found between groups.

Supplementary Figure 4: Acute Latrepirdine (10 min 0.1 nM) pretretment does not affect cytosolic Ca2+ levels during glutamate excitation in neurons

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 acutely with latrepirdine (0.1 nM for 10 min 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 followed by addition of MK 801) 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 acutely latrepirdine (0.1 nM 10 min) pretreated neurons. Vehicle: n = 27 cells; Latrepirdine: (n = 31 cells). Data are shown as mean ± SEM. ns indicates no significant difference was found between groups.

Supplementary References:

1. Freedman JC, Novak TS. Optical measurement of membrane potential in cells, organelles, and vesicles. Methods Enzymol 1989; 172: 102-122.

2. Ward MW, Huber HJ, Weisova P, Dussmann H, Nicholls DG, Prehn JH. Mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate-induced necrosis, apoptosis, and tolerance. J Neurosci 2007; 27(31): 8238-8249.

3. Krohn AJ, Wahlbrink T, Prehn JH. Mitochondrial depolarization is not required for neuronal apoptosis. J Neurosci 1999; 19(17): 7394-7404.