Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer’s disease

doi:10.21203/rs.3.rs-2854082/v1

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Research Article

Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer’s disease

https://doi.org/10.21203/rs.3.rs-2854082/v1

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published 18 Dec, 2023

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Background It has been suggested that exercise training and postbiotic supplement could decelerate the progress of functional and biochemical deterioration in double transgenic mice overexpresses mutated forms of the genes for human amyloid precursor protein (APP^sw) and presenilin 1 (m146L) (APP/PS1^TG). Our earlier published data indicated that the mice performed better than controls on the Morris Maze Test parallel with decreased occurrence of amyloid-β plaques in the hippocampus. We investigated the neuroprotective and therapeutic effects of high-intensity training and postbiotic supplementation.

Methods Thirty-two adult APP/PS1 TG mice were randomly divided into four groups: 1. control, 2. high-intensity training 3. postbiotic, 4. combined (training and postbiotic) treatment for 20 weeks. In this study, the whole hemibrain without hippocampus was used to find molecular traits explaining improved brain function. We applied qualitative RT-PCR for gene expression, Western blot for protein level, and Zymography for LONP1 activity. Disaggregation analysis of Ab-40 was performed in the presence of Lactobacillus acidophilus and Bifidobacterium longum lysate.

Results We found that exercise training decreased Alzheimer’s Disease (AD)-related gene expression (NF-kB) that was not affected by postbiotic treatment. The preparation used for postbiotic treatment is composed of tyndallized Bifidobacterium longum and Lactobacillus acidophilus. Both of the postbiotics effectively disaggregated amyloid-β/Aβ-40 aggregates by chelating Zn2+ and Cu2+ ions. The postbiotic treatment decreased endogenous human APPTG protein expression and mouse APP gene expression in the hemibrains. In addition, the postbiotic treatment elevated mitochondrial LONP1 activity as well.

Conclusion Our findings revealed distinct mechanisms behind improved memory performance in the whole brain: while exercise training modulates NF-kB signaling pathway regulating immune response until postbiotic diminishes APP gene expression, disaggregates pre-existing amyloid-β plaques and activates mitochondrial protein quality control in the region of brain out of hippocampus. Using the above treatments complements and efficiently slows down the development of AD.

Bifidobacterium longum

Lactobacillus acidophilus

Alzheimer’s disease

cognitive function

gut microbiota

mitochondrial protein quality control

metal ion chelation

Alzheimer’s Disease (AD) is currently a non-treatable disease with a gradually increasing incidence causing mild to very sever loss of memory and wide range of associated disorders (1, 2). Because of the lack of efficient pharmacological treatment of AD, there is a huge interest in the possible preventing tools of AD. Evidence is accumulated and showing that regular physical exercise can attenuate the incidence of AD and even lessen the progress of this disease (3–5). Exercise has systemic effects on body showing beneficial, health promoting effects in all organs, despite of the very different oxygen supply, and metabolism of various organs during physical exercise(6). One of the intriguing adaptive response to exercise training involves gut microbiome. The gut-brain axis has been proposed significantly interferes with the function of both organs and more and more data suggest that the microbiome of gut has a powerful effect in gut-brain axis (7, 8). Clinical trial showed that Bifidobacterium bifidum and Bifidobacterium longum containing postbiotics caused changes in gut microbiota and promoted mental flexibility, alleviated stress in healthy older adults, along with causing changes in gut microbiota(9). The microbiome of the gut is crucial for the breakdown of dietary nutrients, regulation of intestinal and systemic immune responses, production of small molecules critical for intestinal metabolism, as well as the generation of several gases that can modulate cellular function. Due to the complex function of the gut microbiome, the diversity of microbes is defined by the number and abundance of the distribution of distinct types of organisms (1).

In our earlier paper we reported that on APP/PS1 transgenic mice exercise training and postbiotic treatment decreased the level of amyloid-β plaques and improved spatial memory assessed by Morris Maze test (10). The result suggested that exercise training and postbiotic treatment have had distinct mode of actions. Our treatment contained tyndallized Bifidobacterium longum and Lactobacillus acidophilus lysates, which could cause the beneficial effects of our treatment. FRAMELIM® contains formerly killed postbiotics, which accordingly to the new nomenclature accepted to call postbiotics (11). Previously it was shown that intracellular cell-free extract of these strains demonstrated strong Fe²⁺, Cu²⁺ chelating ability (12) that was not examined earlier to play a role in modulating development of AD.

It was as well shown that supplementation of Lactobacillus acidophilus attenuated the effects of chronic fatigue and rescued T cell function on athletes (13). The results of another human study suggest that treatment with Bifidobacterium and Lactobacillus decreased the incidence of upper respiratory infection on healthy adults (14). Lactobacillus acidophilus appears to host the surface layer proteins with the molecular weight around 45 kDa, which administration can suppress the activity of myeloperoxidase and TNF-α expression (15). Deletion of gene which encodes lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus resulted in down regulation of IL-12 and TNFα and increased IL-10 levels and suppressed the capability of T-cell activation (16). Our earlier results showed that Bifidobacterium and Lactobacillus treatment decreased Aβ levels (10), and similar data was reported by other paper as well (17), (18). These results encouraged us to study the molecular mechanism behind this attractive effects of Framelin treatment on the progress of AD in transgenic mouse model.

Materials

FRAMELIM® contains tyndallized Bifidobacterium longum and Lactobacillus acidophilus lysates, in addition vitamins A, B1, B3, B6, B9, B12 and omega 3 fatty acids in cod liver oil was included. SIGMAFAST™ protease inhibitor cocktail tablets and PhosSTOP™ phosphatase inhibitor tablets, Human Aβ40, purity ≥ 90% and Thioflavin T was purchased from Sigma Aldrich. Ltd. (Germany). 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO), purity ≥ 95% was from Cayman Chemicals (US). APP rabbit (Abcam - ab101492), human reactive APP mouse ( Biolegend − 6E10 clone), LONP1 mouse ( Proteintech − 66043-1-Ig), SDHA rabbit (SantaCruz - sc-98253), GAPDH mouse (Sigma-Aldrich) antibodies was bought from commercial vendors. mAPP, mNFkB, mSOD-1 primer pairs was ordered from Sigma Aldrich (Germany).

Experimental animals, training procedure and Postbiotic supplementation

Thirty two male APP/PS1 transgenic mice (B6C3-Tg (APPswe,PSEN1dE9)85Dbo/Mmjax; APP/PS1^TG) were randomly assigned to control (APP/PS1^TG-C), exercise (APP/PS1^TG-Ex), postbiotic treated (APP/PS1^TG-Pt) and combined (exercise and postbiotic treated) (APP/PS1^TG-Ex-Pt) groups. For The open field test, The Morris Water Maze Test, the spontaneous alteration tests, and the microbiome investigation, we added wild type mice of similar ages, from the same colony, as absolute controls (Wt). Treatments were carried out for 20 weeks starting at the age of 3 months, with the aim of decreasing the progress of AD development and functional impairment. Interval treadmill running was applied as the exercise regimen for the combined groups. Previously, animals were habituated on a motor driven treadmill (Columbus Inst. Columbus Ohio) at the assigned running speed for two weeks. Training was performed four times a week, for 60 min. Each training session lasted ten cycles, each cycle consisting of four minutes at high-intensity (20 m/min) and two minutes at low intensity (10 m/min). Transgenic control and postbiotic treated groups (APP/PS1^TG-C and Pt) were placed on the treadmill and stayed there for five minutes/day on the stationary belt.

FRAMELIM® was administered five times a week (120 mg/day) for 20 weeks along with rodent chow (SDS /VRF1(P)). Considering that the mouse species has a high vitamin A and B storage capacity and needs a long time (up to 40 weeks) (19) to induce Vitamin A and B deficiency therefore FRAMELIM does not have significant impact on elevation of the vitamin– levels that can influence amyloidosis. We have daily monitored the food uptake of all mice and postbiotic treatment did not alter the amount of food and water intake of animals. After the 20 weeks of treatment, animals were exposed to two weeks of cognitive testing, but even during testing the postbiotic treatments were continued. After the cognitive tests were completed, animals were anesthetized with an intraperitoneal injection of ketamine (Richter, concentration: 100 mg/ml) /xylazine (Produlab Pharma, concentration: 20 mg/ml) cocktail in a dose of 0.1 ml / 10 g bodyweight and transcardially perfused with heparinized ice-cold saline. Brain was removed and measured, rapidly dissected in half along the corpus callosum. One hemibrain was postfixed in 4% paraformaldehyde (PFA) for immunohistological staining that was used for our earlier publication (10). The other hemibrain was dissected into three parts (frontal, parietal and occipital), and the hippocampus was removed. In this study we used whole hemibrain, excluding hippocampus. All parts were collected, frozen in liquid nitrogen and stored at − 80°C until further biochemical analysis.

Preparation of Aβ-40

Aβ-40 and Aβ-42 are the predominant species in the Aβ family, with the former being more abundant and the latter more toxic. Since Aβ-40 contains the same metal binding amino residues (His-6, -14 and − 13) as Aβ-42 and is cheaper, it was used to perform the following experiments. Human Aβ-40 (Sigma Aldrich. Ltd., Germany, ∼ 90%) stock solutions were prepared according to the manufacturer instructions. Lyophilized Aβ-40 was dissolved in Ca²⁺-free Phosphate Buffer Saline at 1 mg/ml (230 µM) and incubated 37 ^oC for 4 days.

Disaggregation assays

Disaggregation analysis of Aβ-40 was performed according to Chen et al. (20) with minor modifications. In disaggregation tests, Aβ-40 Zn²⁺/Cu²⁺ aggregates were prepared by treating fresh Aβ-40 solution with Zn²⁺ or Cu²⁺ and incubating at 37°C for 24 h with constant agitation. Afterwards, DMSO-solved Lactobacillus acidophilus and Bifidobacterium longum lysate, FRAMELIM® and DMSO – as a vehicle control -was added and incubated at 37°C for another 24 h with continuous agitation. The applied dose of Lactobacillus acidophylus and Bifidobacterium longum lysate was 10 µg/ml. In case of FRAMELIM® low (10 µg/ml) and high (100 µg/ml) dose was applied. The high dose of FRAMELIM® contained ∼ 10 µg/ml postbiotic equivalent. To characterize modulation of the aggregation UV/Vis spectrum (wavelength range of 190‒840 nm) was measured as well by DeNOVIX DS-11 FX Spectrophotometer/ Fluorometer before ThT fluorescence assay as well.

Fluorescence of thioflavin T (ThT)

After 2 days of disaggregation assay, DMSO-solved ThT at final concentration of 6 µM was applied to detect beta-amyloid aggregates. The fluorescence was measured after shaking 5 min in 100 ul volume of the incubated sample on ClarioStar plate reader (BMG Laboratories, Germany). Fluorescence spectrum was examined (λ_ex = 415 nm) with an exit slit width of 1 nm between 442 and 600 nm (ClarioStar, Germany). The sample compartment was kept at 21°C.

Mitochondrial, cytosolic and nuclear fraction preparation

Fractionation was performed according to Frezza et al. (21) with minor modifications. Briefly, the frozen brain tissue of each animal was immersed in ice-cold IB_c buffer (10 mM Tris-MOPS, 10 mM Tris-EGTA and 0.2 M Sucrose pH 7.4) in a ddH₂O rinsed, precooled potter glassware, buffer volume was 10-fold. The tissue was homogenized by 3–4 times gentle stroke at 4 ^oC. The homogenate was centrifuged at 600 g, at 4 ^oC for 10 minutes. Supernatant was processed to further separation. The pellet included extracellular matrix elements, tissue debris and nuclear fraction was sonicated and sampled for Western blot. The supernatant was further centrifuged at 7000 g, at 4 ^oC for 10 minutes. The mitochondrial pellet was solved up in IBc buffer and used for gelatine zymography and/or sampled to further Western blot analysis. The initial and leftover supernatant was processed to Western blot sample considering as total and cytosolic fraction. Protein concentration was measured in each fraction using the Bradford assay (22).

Analysis of mitochondrial proteolytic activity analysis through gelatine zymography

Zymography assays were performed according to Kupai et al. (23) with minor alterations. Briefly, the zymography was performed using a 10% SDS-PAGE separation gel with 0.1% of gelatin. Forty micrograms of mitochondria fraction or total lysate from each group was incubated on charging buffer (100 mM Tris pH 6.8, 5% SDS, 20% glycerol, 0.1% bromophenol blue) for 10 min on ice, in a proportion of 1:1 (v/v). After the run, the gels were incubated in renaturation buffer (2.5% Triton X-100) for 30 min, with soft agitation. Then, the zymogram gels were changed to a development buffer (50 mM Tris, 5 mM NaCl, 10 mM CaCl₂, 1 µM ZnCl₂, 0.02% (v/v) Triton X-100, pH 7.4) for more 30 min, also with soft agitation. Finally, the gel immersed into a new development buffer, and incubated overnight at 37°C, in case of mitochondrial proteins the incubation time was 40 hours. For specific inhibition studies zymograms were incubated in the presence of 10 mM EDTA, 10 mM EGTA or 6,4 µM CDDO. The zymography gels were stained with 0.12% (w/v) Coomassie Blue G-250 prepared in 20% methanol, after 1 h fixation in a solution of 10% acetic acid and 40% methanol. Distaining was performed with 25% methanol and stained gels were scanned.

Western blot analysis

The brain tissue of each animal was homogenized in ice and lysed in a lysis buffer containing 137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% Nonidet P-40, 10% glycerol, and tablets of protease and phosphatase inhibitors. Lysates were centrifuged for 15 min at 14.000 g at 4 C. Protein concentration was measured using the Bradford assay (22). Proteins were separated on 8–15% (v/v) SDS-PAGE (sodium dodecyl sulphate–polyacrylamide) gels at room temperature (RT) and transferred onto PVDF membrane (pore size: 0.2 and 0.4 µm) at 4 ^oC. The nonspecific binding of immune-proteins was blocked with 5% BSA (bovine serum albumin) dissolved in Tris-buffered saline, Tween-20 (TBS-T) for 1 h at RT. After blocking, the membranes were incubated with primary mouse and human reactive APP rabbit (1:3000, Abcam - ab101492) and human reactive APP mouse (1:3000, Biolegend − 6E10 clone); LONP1 mouse (1:3000, Proteintech − 66043-1-Ig), SDHA rabbit (1:3000, SantaCruz - sc-98253), NF-kB rabbit (1:3000, Cell Signaling - #3032), GAPDH (mouse, 1:40,000; Sigma-Aldrich) antibodies in TBS-T containing 5% BSA, overnight at 4 C. After overnight incubation the membranes were rinsed in TBS-T attended by one hour of incubation with HRP-conjugated secondary antibodies at RT. The secondary HRP-conjugated antibodies were anti-rabbit, anti-mouse IgG in TBS-T containing 1% BSA (1:10,000; Jackson Immunoresearch). Between incubation times, the membranes were washed repeatedly − 4 times for 10 min - and after the last washing session, incubated with an enhanced chemiluminescent reagent (ECL Star Enhanced Chemiluminescent Substrate; Euroclone) for one minute. The protein bands were visualized on X-ray film. Bands were quantified by ImageJ 1.52 software, and total protein normalization was used for the analysis.

Quantitative Real-Time PCR

Total RNA was extracted from hemi-brain tissue samples by using Nucleo Spin RNA plus RNA isolation kit (Macherey Nagel, Düren, Germany). RNA integrity was tested on ethidium bromide stained 2% agarose gel. RNA purity was measured with DeNovix ds-11 Spectrophotometer (DeNovix, Wilmington, USA) and samples with 260/280 nm absorbance higher than 2 were considered pure and processed forward. RT-cDNA synthesis reaction was performed by Tetro cDNA Synthesis Kit (Bioline London, UK) the total input RNA was 5 µg / reaction, with random hexamer primer amplification according to the manufacture’s protocol. Synthetized cDNA was 10 diluted with PCR grade DEPC-treated water and used for PCR amplification. The mRNA level was PCR quantified by using Corbett Research RG-6000 Real Time PCR Thermocycler (QUIAGEN, USA). IMMomix mastermix (Bioline London, UK) was used. Reverse and forward primers were present at 1 µM concentration with 1% Evagreen. Primer efficiency was accepted at E = 0.90–1,1 respectively, the amplification specificity was checked by melting point analysis. The thermal profile was 50 ^oC for 3 min, 95 ^oC for 10 min, and subsequently 40 cycles of 95 ^oC for 20 s, 60 ^oC for 20 s and 72 ^oC for 20 s. PCR amplification were performed using the following primers: for mouse APP, forward 5’-TGTGCCAGCCAATACCGAA-3’ and reverse 5’-CCAGAACCTGGTCGAGTGGT-3’; for mouse NF-κB, forward 5’-ATGCCGAACTTCTCGGACAG-3’ and reverse 5’-GTGTTTATGGTGCCATGGGTG-3’; for SOD-1, forward 5’-GGAACCATCCACTTCGAG-3’ and reverse 5’-CTGCACTGGTACAGCCTTGT-3’; for mouse β-Actin, forward 5’-AGATCAAGATCATTGCTCCTCCT-3’ and reverse 5’-ACGCAGCTCAGTAACAGT − 3’. The primer sequences shown above were synthesized by Sigma Aldrich (Germany). Results were analysed using the software with relative quantification method and β-Actin as endogenous control was used. Fold changes in each target mRNA expression relative to β-Actin were calculated by Pfaff method and the 2^−ΔΔCT method. Expression of mRNA is defined as the change in mRNA copy numbers relative to APP/PS1 control samples.

Statistical analysis

Prism 5.01 (GraphPad Software) was used for the statistical analysis. Data shown are the mean ± SEM with P < 0.05 considered statistically significant. Two-tailed, unequal variances unpaired t-tests were used for comparisons between two groups applying Welch's t-test. Group differences were analysed with Oneway analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for multiple groups. No statistical methods were used to predetermine sample sizes, but our samples sizes (mouse experiments) are like those reported in previous publications.

Postbiotic supplement disintegrates β-sheet aggregation of Aβ-40

First, we examined whether postbiotic supplementation may influence formation of Aβ-40 plaque. We performed dis-aggregation assay to characterize formation of Aβ-40 plaque in the presence of metal ions and postbiotics. The intensity of Thioflavine (ThT) fluorescence measures the beta-sheet contents of amyloid-ß₄₀ plaque. It increased significantly in the presence of Zn²⁺, however same dose of Cu²⁺ ions resulted in minor elevation of the fluorescence. It suggests Zn²⁺ is more effective in enhancing of Aβ-40 plaque formation (Figure 1A and 1B). Characteristic alterations in UV/Vis spectra were also observable from 190 to 230 nm in the presence both of Zn²⁺ and Cu²⁺ions. Presence of Zn²⁺ ion increased Aβ-40 absorbance except range from 200 to 210 nm (Figure 1C). Intriguingly, presence of Cu²⁺ ion decreased Aβ-40 absorbance ranging from 200 to 210 nm and increased it out of this range (Figure 1D). Based on ThT fluorescence, FRAMELIM^® only at high 100 mg/ml dose inhibited Zn²⁺ion induced formation of Aβ-40 plaque (Figure1A) while Cu²⁺ promoted aggregation was marginally modulated (Figure1B). Based on UV/Vis spectra, FRAMELIM^® only at high dose inhibited Zn²⁺ ion induced elevation of Aβ-40 plaque absorbance (Figure 1C). However, Cu²⁺ ion decreased Aβ-40 absorbance ranging from 200 to 210 nm was counteracted by FRAMELIM^® (Figure1D). Thioflavine (ThT) fluorescence revealed also that both Bifidobacterium longum and Lactobacillus acidophilus lysates effectively diminished Cu²⁺ or Zn²⁺ ions enhanced Aβ-40 plaque formation (Figure1E and 1F). These changes were mirrored in modulation of UV/Vis spectra as well: Zn²⁺ ion induced increase of Aβ-40 absorbance was spectacularly decreased by both postbiotics (Figure 1G). However, modulation of the UV/Vis spectra of amyloid-ß₄₀ in case of Cu²⁺ ion was marginally affected by the postbiotics (Figure 1H). Altogether, it indicates that the applied postbiotics may be an effective constituent of FRAMELIM^® in reduction of amyloid-ß plaques even in vivo.

Postbiotic supplementation decreased human transgene APP-level

Because of metal ion chelating property of postbiotics, it is plausible assumption that it can influence expression and transcription of full-length human APP^TG. Therefore we measured both total (endogenous mouse and human) and human APP levels by Western blot (Figure 2 A,D). The analysis with human reactive anti-Amyloid-β antibody (clone 6E10) exposed that postbiotic supplementation decreased human APP^TG-level, but exercise training of postbiotic supplementation neutralized the observed advantageous decrease (*P<0.05; Figure 2C, 2D). As regard, the endogenous mouse APP and human APP^TG forming together total APP-level was clearly elevated in all APP/PS1^TG mice. We were unable to detect significant differences among groups at protein level (Figure 2A, 2B).

Postbiotic supplementation and exercise training modulate expressions of AD-related genes

Because of inflammation induced oxidative stress plays a crucial role in development of AD, we studied mRNA levels of some relevant transcription factor and protein. Presence of APP/PS1 transgene results in elevated total mouse APP mRNA-level in all APP/PS1 related examined groups. However, postbiotic supplement decreased APP-level comparing to control (P<0.05, Fig.3). Exercise training decreased significantly NF-κB (*P<0.05; Fig.3) and SOD1 (*P<0.05; Fig.3) mRNA-levels. Cotreatment with postbiotic supplement significantly inhibited expression of NF-κB (*P<0.05; Fig.3) but not in the case of SOD1 expression (*P<0.05; Fig.3).

Postbiotic supplementation and exercise training elevated mitochondrial level of LONP1 and SDHA

Analysis of mitochondrial proteins revealed that postbiotic supplement and exercise training changed mitochondrial composition. Postbiotic supplement increased mitochondrial matrix localized ATP-dependent protease, LONP1-level significantly. Exercise training of postbiotic supplement resulted in slightly decreased but still significant LONP1 level increment (*P < 0.05; Figure 4A and 4B). Postbiotic supplement increased mitochondrial SDHA-level as well and exercise training of postbiotic supplement resulted in slightly decreased but still significant elevation (*P < 0.05; Figure 4A and 4B). Level of NDUFV1 that is hydrophilic protein firmly attached to mitochondrial matrix remained constant in all groups. Coomassie staining of PVDF membrane proved equal loads of mitochondrial proteins (Figure 4A and 4B). Change in Lon protein levels and activity does not always correlate, because post-translational regulation of LONP1 activity (24).

Proteolytic activity analysis through gelatine zymography (25) revealed that postbiotic supplementation resulted in a few gelatinase activities. Light minor bands are observable at ~ 250 and ~ 150 kDa, defined bands at ~ 100 and ~18 kDa. The maximal activity at 100 kDa that is the estimated molecular weight of monomer LONP1 (black asterisks, Figure 5A).

Postbiotic treatment resulted in 10-fold increase in the activity comparing to untreated wild type and APP/PS1 mice control. Exercise training resulted in weak not significant elevation of gelatinase activities at ~100 kDa. Intriguingly co-treatment resulted in no gelatinase activity at all, suggesting that exercise training somehow counter-acted postbiotics induced LONP1 activity (Figure 5A, Figure 5B). To prove that the observed postbiotic supplement induced gelatinase activities exclusively attributable to LONP1, few vital factors of the activity were studied. Inhibition of the activity resulted by ATP deprivation; by chelation of Ca²⁺ and Mg²⁺ ions with EGTA and EDTA; by specific LONP1 inhibitor CDDO, that significantly decreased the activity (Figure 5C). These data suggest postbiotic supplement resulted in both elevated LONP1 mitochondrial level and activity.

There is a huge need to identify natural and pharmacological interventions that can prevent, attenuate or slow down the progress of AD. Here we report that both Bifidobacterium longum and Lactobacillus acidophilus lysates effectively reduces aggregation of Aβ−40 induced by either Cu²⁺ or Zn²⁺ in vitro and in vivo models. The role of metal ions, Cu²⁺, and Zn²⁺ has been suggested to play a crucial role in changing the chemical architecture of Aβ−40/42 in AD and increasing the toxicity (26–29). Cu²⁺ and Zn²⁺ can hypermetallate Aβ−40/42 resulting in production of H₂O₂ and oxidative stress (30). It was even suggested that the greater incidence of AD among females, could be at least a part, dependent on the higher activity of Zn transporter ZnT3 and suppressed ability to chelate metal ions (30). We have already shown that supplementation of FRAMELIM® containing Bifidobacterium longum and Lactobacillus acidophilus decreased the accumulation of Aβ−40 aggregates and toxicity can be reduced (10), and here we shown that was at least partly mediated by chelation of Cu²⁺, and Zn²⁺.

Inflammation is a co-existing and/or causative process of AD (31–34). Transcription factors like SP1 and NF-kB are mediators of inflammatory process and downregulation of these transcription factors not just suppress inflammation but decrease production of reactive oxygen species (ROS) as well (4). Here we confirmed that exercise especially with postbiotic treatment decrease the involvement of NF-kB (35, 36) in inflammation and our data showed that in APP/PS1 model this process has neuroprotective role (10).

We have earlier shown that the mitochondrial house-keeping enzyme, the LONP1 is sensitive to exercise stimulus and exercise induction is associated with decreased accumulation of damaged mitochondrial proteins in skeletal muscle and increased endurance capacity (37–39). Here, our data revealed that exercise training and Postbiotic treatments enhanced LONP1 levels and activity, assessed by gelatine zymography. LONP1 is responsible of removal of damaged proteins in the mitochondria, and because of excessive generation of ROS in this organelle this has a vital role in cell survival. AD is known to associated with increased oxidative stress (40, 41), therefore the present novel observation, that exercise and postbiotic increase LONP-1 level and activity adds an important new mosaic to the better understanding of exercise and postbiotic treatments on AD.

The results of the present study revealed that postbiotics treatments and exercise training have beneficial effects on APP/PS1^TG mouse model for Alzheimer’s Disease, which are partly mediated by the chelating effects of heat-inactivated Bifidobacterium longum and Lactobacillus acidophilus on both Zn²⁺ and Cu²⁺ ions. Moreover, the combined effects of exercise and postbiotics enhance antioxidant system, supress inflammation and increase the mitochondrial housekeeping via activation of LONP1. These results give a hint that by mild training protocol in combination with the above postbiotics may offer more effective treatment protocol for AD.

Ethics approval and consent to participate

The investigation was performed according to the requirements of “The Guiding Principles for Care and Use of Animals, EU”, and was approved by the Semmelweis University Ethics Committee (No: PEI/001/2105–6/2014).

Consent for publication

Not applicable

Availability of data and materials

All data generated or analysed during this study are included in this published article and supplementary file.

Competing interests

The authors declare that they have no competing interests. FRAMELIM, which qualified as a special food after professional clinical tests, was formulated based on Janos Feher’s former inventions.

Funding

This study was supported by OTKA (112810 and National Excellence Program (126823) grants awarded to Zsolt Radák beside it was supported as well by TEKA (Hungarian University of Sports Science Grant, 2022) grant awarded to Attila Kolonics.

Authors' contributions

Zoltán Bori, Attila Kolonics, Dóra Ábrahám, Ferenc Torma performed the analysis of samples. Attila Kolonics, János Fehér, Zsolt Radák participated in the research design and the drafting, writing and revisions of the manuscript. All authors have read and approved the final version of the manuscript and agree with the order of presentation of the authors.

Acknowledgements

Authors acknowledge the assistance of Professor A.W. Taylor in the preparation of the manuscript.

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No competing interests reported.

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published 18 Dec, 2023

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Editorial decision: Major revision
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Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer’s disease

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Abstract

Figures

Introduction

Materials and Methods

Materials

Experimental animals, training procedure and Postbiotic supplementation

Preparation of Aβ-40

Disaggregation assays

Fluorescence of thioflavin T (ThT)

Mitochondrial, cytosolic and nuclear fraction preparation

Analysis of mitochondrial proteolytic activity analysis through gelatine zymography

Western blot analysis

Quantitative Real-Time PCR

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1