Biological and biophysics aspects of metformin-induced effects: cortex mitochondrial dysfunction and promotion of toxic amyloid pre-fibrillar aggregates

Metformin is a fluorescent molecule able to reach the brain.

Absorption or fluorescence phenomena of ultraviolet or visible lights by a molecule depend on electron transitions between molecular orbital energy levels. Due to the presence of two double conjugated bonds the possibility of fluorescence phenomenon in metformin has been investigated. The emission spectrum obtained by fluorescence measurements indicates that metformin, once excited at 395 or 488 nm, has the emission peaks at 475 and 520 nm, respectively (data not shown). This property allows analyzing the presence of metformin into the brain of mice after its dispersion in drinking water. Metformin was administered to C57B6/J mice and after seven days the mice were sacrificed and the brains analyzed by using an imaging instrument. A strong signal was detected in the treated mice whereas no signal was found in the control, thus indicating that metformin has reached the brain (Fig. 1a,b). The delivery of metformin to the brain was confirmed by the increased levels of phosphorylated AMP-activated kinase (AMPK), one of the main molecular target of metformin (Fig. 1c,d). Thus, metformin crosses the blood brain barrier (BBB) and has an impact on the brain biochemical pathways.

Chronic metformin treatment stimulates APP processing mainly in brain cortex region

To ascertain the possibility that metformin is a risk factor for AD onset, especially in long term administration, C57B6/J mice were treated with metformin for seven days or three months. After these treatments proteins extracted from cortex and hippocampus, two-brain area mainly damaged in AD, were submitted to Western blot. Changes in the levels of BACE1, an enzyme required for APP processing to produce Aβ, its pathogenic cleavage product, and the same APP were measured. Activation of AMPK was evaluated for confirming the biochemical activity of metformin in the brain (Fig. 2). An increase of the levels of BACE1 and APP expression was detected in the cortex after seven days of treatment (Fig. 2a). In contrast, an increase of BACE1, and a decrease of APP were found after chronic treatment in the cortex, suggesting that an enhanced processing may be occurred (Fig. 2c). Furthermore, after both treatments, no significant differences in BACE1 and APP expression in the hippocampus were detected (Fig. 2b, d). To validate the hypothesis that the decreased presence of APP in the cortex was a consequence of the β-secretase increased activity, a quantitative real-time PCR (qRT-PCR) experiment was performed. No significant change was observed in APP transcript, strongly signifying that the produced protein was quickly processed (Fig. 2e).

Metformin induces accumulation of Aβ aggregates mainly in brain cortex region

Since the obtained results after three months of treatment suggested an augmented processing of APP mainly in the cortex, we explored the possibility that an increase of Aβ production could have enhanced its aggregation and deposition in the extracellular area. By immunofluorescence analysis, using coronary brain sections, in which cerebral cortex and hippocampus were visible, and anti-APP antibody we observed a diffuse staining around the nuclei of the control and a punctate staining around the cells of treated cortex, suggesting presence of aggregates due to increased processing (Fig. 3a). However, this result was confirmed by staining with Thioflavin T (Fig. 3b), a dye used to reveal the presence of β-sheet protein aggregates because of the increase of its fluorescence emission intensity upon binding to the linear array of β-strand aggregates [38,39]. In particular, we observed aggregates with a dimension ranging below 1 μm mainly in the cortex (Fig. 3b). In contrast, no significant immunoreactivity, or presence of Aβ aggregates, was detectable in hippocampus (Fig. 3a, b).

Metformin induces mitochondrial dysfunction by impairing MPT pores and membrane channels.

Specific mitochondrial proteins involved in distinctive structures regulate cross talk and transport of proteins and metabolites between cytoplasm and mitochondria. We addressed the question if metformin treatment might contribute to the alteration of the components of mitochondrial machinery. After three months of treatment changes in levels of expression were analyzed for some proteins involved in mitochondrial transport of different molecules and markers of mitochondrial dysfunction such as TOM40, VDAC1 and HKI. Western blot of proteins extracted from both cortex and hippocampus of mice treated with metformin and controls were incubated with antibodies against TOM40, VDAC1 and HKI. In agreement with previous data, Western blot analysis showed changes in the levels of expression of the analyzed proteins only in the cortex region. An increase of TOM40 was detected indicating an impairing of MPT pores (Fig. 4a). Further, an increase of total VDAC1 levels were observed in cortex where, in particular, we found together with VDAC1 monomers some dimers and trimers, suggesting that a fraction of the VDAC1 in the membrane is organized in oligomers (Fig. 4c). Moreover, the lower band, indicated by the red arrow in Figure 4C, represents a monomeric species whose electrophoretic mobility is modified by the intramolecular crosslinking of the VDAC1 N-terminal domain. A decrease of HKI amount was also detected suggesting that probably a detachment from VDAC1 was occurred (Fig. 4e). TOM40, VDAC1 and HKI levels were not significantly affected in the hippocampus brain area (Fig. 4b, d, f).

Metformin induces neuronal apoptosis

To assess whether, after chronic metformin treatment, apoptotic cell death occurred in vivo, presence of fragmented DNA was examined in cortex and hippocampus brain sections by the terminal deoxynucleotidyl transferase-mediated, dUTP, nick end labeling (TUNEL) method. TUNEL-positive cells in brain sections of metformin treated mice were markedly higher in cortical region than in hippocampal region or control (Fig. 5a, b).

Metformin directly interacts with Aß peptide influencing its aggregation kinetics in vitro.

In order to assess whether metformin is also able to directly interact with Aβ peptide influencing its aggregation process we performed the fibrillogenesis kinetics by ThT assay. Figure 6 shows the time course of the ThT signal during the fibrillation kinetics of 50 µM Aβ incubated at 37°C in the absence and in the presence of 2mM metformin. The kinetics followed the typical nucleation-polymerization process, described by the sigmoidal profile [36]. In the presence of metformin, the lag phase increased and the amount of final fibrils is reduced in comparison with control. Metformin addition seemed to reduce the effective Aβ concentration leading to initial nuclei, thus interfering with lag-phase.

This result was confirmed by circular dichroism (CD) experiments. CD spectra are sensitive to the secondary structure variation accompanying amyloid cross-β-structure formation during fibrillogenesis. In the initial phase of the aggregation process, the Aβ CD spectrum presents a single minimum that, typical of a random coil structure, is around 200 nm. The minimum shifts towards higher wavelength during amyloid formation. Our results have shown that the presence of 2mM of metformin delayed the β-sheet structure formation of a 50 μM Aβ_1-40 solution. In fact, while the control sample reached the end point of the aggregation reaction (Fig. 7a) after 0.5 h, no significant changes were observed in the presence of the drug in the same time interval indicating that the peptide retained its disordered structure (Fig. 7b), in fully agreement with the fluorescence data. Always in accordance with ThT assay, after 2 h both the samples completed their conversion.

The reduced amount of Aβ_1-40 fibrils detected by ThT assay in the presence of metformin was confirmed by AFM measurements. In Figure 8a-d the morphology of the aggregates formed at the end of the kinetics in the absence and in the presence of metformin are reported. Amyloid fibrils result reduced in number and length when metformin is added in solution. Moreover, prefibrillar oligomeric aggregates are observed at the end of the kinetics of the peptide incubated with the drug.

The characterization of the action of metformin on the aggregation process of Aβ_1-40 peptide was followed by Dynamic Light Scattering (DLS) experiments. Although not specifically focused on chemico-physical features characterizing amyloid formation, like conversion to cross-β-structure, the addition of DLS to other biophysical techniques provides important quantitative information on the hydrodynamic size variation occurring during an aggregation process. In this sense, it represents a suitable technique to monitor the influence of an exogenous molecule on the protein assembly, and, therefore, it is often used for testing drugs for therapeutic purposes. Moreover, light scattering technique does not need the use of extrinsic probes, whose evaluation of their potential influence on the process under study requires high carefulness [40]. Figures 8e and f show the hydrodynamic diameter D_H distribution functions of a sample of Aβ_1-40 peptide undergone to the amyloid formation protocol (37°C and 200 rpm under stirring) with and without metformin, respectively after 0.5 h from the beginning of the process and at the end of the experiment observation (2 h). The increase in ThT assay (Fig. 6) and variation in CD spectra (Fig. 7) reveal that, at that time, in the absence of metformin a significant amyloid cross-β-structure has already formed. Correspondently, two size distributions are revealed by DLS analysis: one centered at around 300 nm and the other one corresponding to larger species of the order of microns. At the same time, in the presence of metformin this higher size species is not formed and only the appearance of species at around 300 nm occurs.

The size distribution for samples collected at the end of aggregation kinetics reveals for the Aβ_1-40 sample grown in the absence of metformin, a marked shift towards higher values in comparison to the initial time, probably corresponding to mature fibril formation. In contrast, only a little increase in the average hydrodynamic diameter (from 300 to 600 nm) in the correspondent sample incubated with the drug is detectable. The overall results from biophysical techniques converge in indicating that the metformin delays amyloid aggregation of Aβ_1-40 peptide, reduces the amount of mature fibrils formed and, more important, stabilizes prefibrillar oligomeric species.

Mice

The experimental procedures employed in the present study were in accordance with the Italian D.L. no. 116 of 27 January 1992 and subsequent variations and the recommendations of the European Economic Community (86/609/ECC). The studies were approved by Ministero della Sanità (Rome, Italy). Male C57BL/6J (B6) mice, purchased from Harlan Laboratories (San Pietro al Natisone Udine, Italy) at 4 weeks of age, were housed under standard conditions of light (12 h light: 12 h darkness cycle) and temperature (22–24 °C), with free access to water and food. After acclimatization (1 week), the animals were divided in two groups (n=10 per group), control and metformin treated. Mice were provided food and received metformin in drinking water (2 mg/mL) for 7 days or three months. After this treatment, animals were sacrificed by cervical dislocation and the brain of age-matched animals were immediately exported and processed for subsequent analysis, as previously described [7]. When necessary, hippocampus and cortex were separated. Biodistribution of metformin in ex vivo brain was detected by using Biospace Lab Imaging Instrument.

Total protein extraction and Western blotting

Brain of mice were homogenized in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, 1mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton, 0.1%SDS, and 0.5% deoxycholate) with protease inhibitors (Amersham) and phosphatase inhibitor cocktail II and III (SIGMA). To remove insoluble material, tissue lysates were sonicated and centrifuged (14,000 rpm, at 4°C, for 30 min). Proteins (50 μg) were resolved by 10% SDS-PAGE gel and transferred onto nitrocellulose filters for Western blotting using anti-APP (1:1000), anti-BACE1 (1:1000) anti-phospho-AMPK (1:1000), anti-TOM40 (1:1000), anti-VDAC1 (1:1000), and anti-HKI (1:1000) purchased from Santa Cruz, anti-β-actin (1:5000) purchased from Sigma. Secondary antibodies conjugated to horseradish peroxidase (1:2000) purchased from Cell Signaling were detected using the NOVEX® ECL HRP chemiluminescence kit (Cat. n° WP20005, Invitrogen) according to the manufacturer’s instructions. In some instances, antibodies were stripped from blots with Restore Western Blot Stripping Buffer (Thermo Scientific) for 10 minutes at room temperature, for antibody reprobing. Band intensities were analyzed with a gel documentation system (BioRad), expression was normalized with β-actin expression. The protein levels were expressed as densitometry and percentage of controls.

Immunofluorescence

For immunofluorescence the brains (n=5 per group) were embedded in paraffin as previously described [7] and coronally sectioned (5µm) using a microtome. Brain sections including the cerebral cortex and the hippocampus were mounted on slides and deparaffinized in xylene solution. Then, the slides were hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 minutes each. After washing in water and PBS the slides were incubated with 3% BSA⁄PBS for 1 h. Next, the sections were incubated with anti-APP (1:50) (Santa Cruz) at 4 °C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1:500; SIGMA). For nuclear staining, the sections were incubated with Hoechst 33258 (5μg/ml) for 20 minutes. After washing in PBS the slides were mounted with cover slips and images were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20X magnification.

Thioflavin T staining

For Thioflavin-T (ThT) staining the brain sections including the cerebral cortex and the hippocampus were mounted on slides The slides were deparaffinized in xylene solution and hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 minutes each. After washing in water, the sections were incubated in filtered 1% aqueous ThT solution for 8 minutes at room temperature. The slides were then dehydrated in ethanol 80% and 95%, for 5 minutes each. After washing in water the slides were mounted with cover slips and the images were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20x magnification.

TUNEL assay

Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL)-positive apoptotic nuclei were detected in brain paraffin sections using an in situ cell death detection kit (Promega) according to manufacturer’s instructions. The number of apoptotic cells was counted in randomly selected fields to calculate the ratio of apoptotic cell per brain area.

Quantitative Real-Time PCR

Total RNA was extracted using RNEasy Mini Kit (Qiagen). Two ng of RNA was used to synthesize the first strand cDNA using RT First-Strand kit (Qiagen). Synthesized cDNAs were amplified using RT² SYBR Green/ROX qPCR Mastermix (Qiagen) and StepOne Real-Time instrument (Applied Biosystem). Gene expression validation was performed using RT2 qPCR Primer Assay for human APP, Presenilin1, β-actin (SABiosciences). Gene expression was normalized to β-actin.

Statistical analysis

All experiments were repeated at least three times and each experiment was performed in triplicate. The results are presented as mean + SD. A one-way ANOVA was performed, followed by Dunnet's post hoc test for analysis of significance. Results with a p-value <0.05 were considered statistically significant, *P < 0.05, **P<0.02.

Sample preparation for biophysics experiments

The synthetic peptide Aβ_1–40 (Anaspec) was pretreated according to the procedure of Fezoui et al. [53] for improving the reliability of experiments at neutral pH. Stock aliquots (200 μg each) were stored at -80°C. Metformin was purchased from Sigma Aldrich. Aβ_1–40 samples were prepared by dissolving the lyophilized peptide in 50 mM phosphate buffer, pH 7.4, at a concentration of about 70 μM. The solution was filtered through 0.22 μm and 20nm filters into a fluorescence quartz cuvette containing a small magnetic stirring bar. Aβ_1–40 concentration was determined by tyrosine absorption at 276 nm using an extinction coefficient of 1390 cm⁻¹M⁻¹. The sample was then diluted to the working concentration of 50 μM by adding the appropriate amount of buffer, concentrated solution of ThT (1 mM), and concentrated solution of metformin (20 mM) when required.

Final samples containing Aβ and metformin were obtained by appropriate aseptically mixing of the protein solutions and placed in closed cuvettes in a cold room at 4 °C, before incubation at higher temperatures. The aggregation kinetics were followed at controlled temperature (37 °C) and under controlled stirring (200 rpm) for 24 hours.

ThT Spectrofluorometric measurements

ThT fluorescence emission was monitored by using a JASCO FP-6500 spectrometer. The excitation and emission wavelengths were 450 and 485 nm, respectively, with 3 nm slit width. ThT concentration was 12 μM. The sample was placed at 37°C in the thermostated cell compartment (10 mm). When required, a magnetic stirrer at 200 rpm (mod. 300, Rank Brothers Ltd., Cambridge) was used.

Control fluorimetric experiments between Thioflavin T and metformin with the purpose of excluding undesired pitfalls due to potential interaction between the dye and the molecule [54] have been performed under the same conditions of aggregation kinetics (37 °C, under stirring).

Circular dichroism spectroscopy

CD measurements were acquired by using a JASCO J-815 CD Spectrometer. Particularly, during the aggregation kinetics, withdrawals of samples at appropriate time were observed. Spectra were recorded at 20 °C using a quartz cell with 0.2 mm path length. Each spectrum measurement was obtained by averaging over eight scans and subtracting the blank solvent contribution.

Dynamic light scattering

The aggregation of Aβ peptide in the presence or absence of metformin was investigated by Dynamic Light Scattering. The samples were placed into a dust-free quartz cell without further filtering and kept at 37°C in the thermostatic cell compartment of a Brookhaven Instruments BI200-SM goniometer. The temperature was controlled within 0.1 °C using a thermostatic recirculating bath. The light scattered intensity and its autocorrelation function were measured at θ = 90° by using a Brookhaven BI-9000 correlator and a 50 mW He–Ne laser tuned at a wavelength λ = 632.8 nm. Due to their Brownian motion, particles moving in solution give rise to fluctuations in the intensity of the scattered light [55,56]. The autocorrelator measures the homodyne intensity–intensity correlation function that, for a Gaussian distribution of the intensity profile of the scattered light, is related to the electric field correlation function:

$g^{\left(2\right)}\left(q,t\right)=[A+B{g}^{\left(1\right)}\left(q, t\right){]}^{ 2}$(1)

where A and B are the experimental baseline and the optical constant, respectively. For polydisperse particles, g⁽¹⁾(q,t) is given by:

$g^{\left(1\right)}\left(q,t\right)= {\int }_0^{\infty }G \left(\mathrm{\Gamma }\right)exp\left(-\mathrm{\Gamma }t\right)d\mathrm{\Gamma }$(2)

Here, G(Γ) is the normalized number distribution function for the decay constant Γ = q²D_T, where q=(4πn/λ)sin(θ/2) is the scattering vector defining the spatial resolution with n and D_T being the solvent refractive index and the translational diffusion coefficient, respectively. The hydrodynamic diameter D_H is calculated from D_T through the Stokes–Einstein relationship:

$D_{T }= \frac{k_{B}T}{3\pi \eta D_H}$(3)

where k_B is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. Intensity-weighted distribution functions P_I of the z-average hydrodynamic diameter D_H were obtained by the analysis of the intensity autocorrelation functions were analyzed by means of a CONTIN-like smoothing-constrained regularization method [57].

Atomic Force Microscopy (AFM)

AFM measurements were performed by using a Nanowizard III (JPK Instruments, Germany) mounted on an Axio Observer D1 (Carl Zeiss, Germany) or on an Eclips Ti (Nikon, Japan) inverted optical microscope. Aliquots of protein solutions were deposited onto freshly cleaved mica surfaces (Agar Scientific, Assing, Italy) and incubated for up to 20 min before rinsing with deionized water and drying under a low pressure nitrogen flow. Imaging of the protein was carried out in intermittent contact mode in air by using NCHR silicon cantilever (Nanoworld, Switzerland) with nominal spring constant ranging from 21 to 78 N/m and typical resonance frequency ranging from 250 to 390 kHz.