Effects of the probiotic formulation SLAB51 in in vitro and in vivo Parkinson’s disease models

In vitro

To investigate if the probiotic formulation SLAB51 contained neuroprotective components, an in vitro model of PD was developed, and neuroprotective and neuronal death pathways were analyzed. The induction of the dopaminergic phenotype of SH-SY5Y neuroblastoma cells, described in the Methods section, was analyzed by contrast phase microscopy, Tyrosine Hydroxylase (TH) expression, dopamine production and immunofluorescence (Figure 1). It is possible to observe that, upon retinoic acid (RA)/phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, cells displayed neuronal morphology, expressed higher levels of TH, produced dopamine and expressed neuronal markers, such as β-tubulin III and growth associated protein-43 (GAP-43). 6-hydroxydopamine (6-OHDA) concentration able to induce a significant decrease of cell viability was evaluated by MTS assay (not shown). On this basis, 35 μM 6-OHDA was chosen for the subsequent experiments, since a 50% mortality with this dosage was obtained.

SLAB51 lysates do not shown toxic effect, as demonstrated by cell viability test on differentiated SH-SY5Y; thus, basing on these preliminary results, 0.1 mg/ml of extract was set as testing concentration for the subsequent experiments. In Figure 2, the MTS assay of cells treated with 35 μM 6-OHDA and SLAB51 is shown. As evident, 6-OHDA led to 50% mortality, while SLAB51 was able to counteract 6-OHDA-induced injury and to restore control conditions.

Since it is worth noting that BDNF pathway is involved in neuroprotection and neuronal survival, we first analyzed, by Western blotting, the protein levels of mature BDNF (mBDNF), phosphorylated tyrosine receptor kinase B (p-TrkB), phosphorylated cAMP response element-binding protein (p-CREB) and phosphorylated extracellular-signal-regulated kinase 5 (p-ERK5). In the presence of 6-OHDA, mBDNF and its specific receptor TrkB as well the survival kinase ERK5 were significantly decreased with respect to control neurons, while the presence of SLAB51 restored the control levels (Figure 3), suggesting a protective action exerted by the probiotic. Further, p-CREB, which is known to control mBDNF levels and the survival pathway phosphoinositide 3-kinase (PI3K)/ phosphorylated protein kinase B (p-Akt), was dramatically decreased upon 6-OHDA and restored at control levels by SLAB51 (Figure 3).

Finally, PI3K/Akt pathway, which is involved in neuronal survival and CREB phosphorylation, as well as the postsynaptic density protein 95 (PSD95) appeared strongly downregulated by 6-OHDA, while the presence of SLAB51 counteracted this effect (Figure 4), thus suggesting that the lysate is able to ameliorate the neuronal synaptic plasticity as well the neuronal survival.

The neuronal death pathway, comprising pro-BDNF, phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal–regulated kinase (p-ERK1,2) and P75 was then analyzed. All these proteins were significantly increased by 6-OHDA, while the presence of SLAB51 restored the control conditions, thus confirming a neuroprotective role exerted by the probiotic (Figure 5).

Recent evidence reported the involvement of activated peroxisome proliferator activated receptor γ (PPARγ) in modulating BDNF levels in different pathologies, including PD [30]. Thus, on light of the results collected so far, we assayed PPARγ level through Western blotting analysis. Indeed, in 6-OHDA-treated cells a significant reduction of the transcription factor was apparent; while SLAB51 lysate increased PPARγ protein levels (Figure 6). Further, since it is known that a product of lipid peroxidation, 4-hydroxynonenal (4-HNE) is generally increased during oxidative stress, as occur in neurodegeneration processes, and to highlight a potential role of probiotics in counteracting 6-OHDA oxidative injury, 4-HNE protein adducts by Western blotting in in vitro samples were analyzed. It is possible to observe that the probiotic formulation significantly reduced the level of 4-HNE proteins adducts, thus suggesting a protective role of SLAB51 against oxidative damage (Figure 6). Once obtained the above promising results in vitro, in order to evaluate if this formulation was able to modulate neuroprotective pathways in a more complex model, we tested the probiotic in unilateral 6-OHDA-lesioned animal model.

In vivo

To test the formulation in vivo, SLAB51 diluted in drinking water was daily administered via oral gavage for 2 weeks previous 6-OHDA injection and followed for further 3 weeks (as indicated in “Material and Methods” section and in the timeline Figure 7A, total 5 weeks). Oral administration of the probiotic formulation did not generate neither mortality nor significant variances in the average body weights, in control and treated mice (Figure 7B). In Figure 7B also behavioral tests are shown. Notably, the cylinder test, considered to evaluate locomotor asymmetry in rodent models of CNS diseases, showed that striatum lesion induced a robust and significative decrease in mice motor performance, in particular, it was possible to appreciate a decrease of the use of contralateral paw. Interestingly, SLAB51 was able to counteract behavioral impairment induced by 6-OHDA inoculation, restoring the control conditions.

The Elevated Body Swing Test (EBST), index of asymmetrical motor performance of hemi-parkinsonian models in a drug-free state, in the same figure is shown. 6-OHDA-lesioned mice showed right-biased swings of 70% or greater respect to control animals, while probiotic formulation treatment was able to counteract this effect, in fact, at 2 weeks the animals showed the same behavior of SHAM group. These findings were confirmed by apomorphine test, a tool to monitor motor impairment and functional improvement [31]. In particular, the dopamine (DA) receptor agonist apomorphine (APO), acting post-synaptically and hyperstimulating supersensitive DA receptors in the denervated striatum, leads the animal to rotate in the opposite contralateral direction, i.e., away from the lesioned side. Indeed, in our experimental conditions, upon 6-OHDA mice showed increased contralateral rotations, while the 6-OHDA mice treated with the probiotic formulation had a similar behavior to SHAM groups (Figure 7).

Further, the immunostaining of TH in dopaminergic neurons was performed; it was possible to observe in 6-OHDA-treated animal a marked decrease of TH immunoreactivity, while SLAB51 rescued dopaminergic neurons in both substantia nigra and striatum (CPu) (Figure 8). To further validate these data, immunofluorescence analyses for Dopaminergic Transporter (DAT) in mice substantia nigra were performed. As reported in Figure 9, a massive reduction of DAT fluorescence intensity was observed upon 6-OHDA inoculation (in the right side), while the probiotic formulation counteracted this effect, thus suggesting that SLAB51 exerted neuroprotective activities.

Recent studies have demonstrated neuroinflammation and microglia activation in PD [30]. For this reason, immunofluorescence analyses and quantification for the specific marker of microglial activation, microglia ionized calcium-binding adapter molecule 1 (Iba1), and for astrogliosis, glial fibrillary acid protein (GFAP), in brain slices were performed (Figure 10). It is possible to observe a significant increase of Iba1 and GFAP fluorescence intensity in 6-OHDA slices, while SLAB51 was able to counteract 6-OHDA-induced effects, thus indicating a reduction in neuroinflammation.

On this basis, PPARγ, a ligand-dependent transcription factor involved in neuroinflammation, oxidative stress and energetic metabolism, that is also able to stimulate neurotrophins release (including BDNF) [30], was analyzed by immunofluorescence in brain slices (Figure 11). Interestingly, SLAB51-treated samples showed the transcription factor at nuclear level, while in 6-OHDA-treated animals, the fluorescence intensity for PPARγ was strongly decreased and, further, localized at cytoplasmic level. Western blotting analyses for PPARγ (Figure 12) demonstrated a restoration of this protein levels in 6-OHDA/SLAB51 treated animals to those of control group. Western blotting results, concomitant with PPARγ localized into the nucleus, may suggest that its activation could be responsible for the reduced neuroinflammation and for the neuroprotection via BDNF pathway, as confirmed by Western Blot analysis performed on in vivo samples. Indeed, BDNF and its receptor TrkB showed the same behavior of PPARγ, suggesting that SLAB51 was able to counteract the toxin-induced lesion both in substantia nigra and striatum (Figure 12).

Recent findings indicated that hemeoxygenase-1 (HO-1) is regulated by upstream regulators of PPARγ [32]. The antioxidant enzyme HO-1 with established cytoprotective effects has been demonstrated to modulate several pathological processes, including PD. Notably, HO-1 is involved in the release of neurotrophic factors, in the sustainment of dopaminergic neuronal survival in substantia nigra, and in preventing α-synuclein aggregation [33]. Interestingly, in our experimental conditions, 6-OHDA significantly decreased HO-1, while the probiotic formulation was able to counteract 6-OHDA effects, reverting the levels of HO-1 to those of control condition as shown in Figure 13.

In addition, nuclear transcription factor-erythroid 2 related factor (Nrf2) is able to bind the antioxidant response element (ARE) present in the HO-1 promoter region [34]. Nrf2 activity decreases with aging and represent one of the main PD risk factors, effectively, the increase of Nrf2 provides protection to dopaminergic neurons by counteracting oxidative stress injury [35]. As shown in Figure 13, p-Nrf2 (transcriptionally active) protein levels were significantly downregulated in 6-OHDA-injured animals, while SLAB51 was able to counteract 6-OHDA effects, both in substantia nigra and striatum.

Further, NRF2-ARE system interacts with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), a protein complex involved in cell survival and cytokine release, related to neurodegenerative and neuroinflammatory conditions. In fact, in our experimental conditions, NF-ĸB protein levels were upregulated upon 6-OHDA challenge, while the probiotic treatment reverted this protein to control conditions, both in substantia nigra and striatum (Figure 13), thus suggesting a control in neuronal inflammation and the immune response.

Finally, to confirm the potential pro-survival effect of SLAB51, the apoptosis promotion in dopaminergic neurons of substantia nigra was analyzed by TUNEL assay. As reported in Figure 14, apoptosis dramatically increased in 6-OHDA treatment, while the presence of the probiotic mixture reduced apoptotic nuclei index (to about 10%), thus suggesting that the probiotic formulation protects against 6-OHDA-mediated apoptosis.

In vitro experiments

Preparation of SLAB51 bacterial lysates

Amount of 1 g of SLAB51 formulation (sold as Sivomixx, Mendes, Switzerland) has been suspended in 10 ml of Phosphate Buffer Saline (PBS, Euroclone, UK) for bacterial lysates preparation, subjected to centrifuge and sonication processes and at the final step the lysates were also filtrated to remove whole bacteria remaining as previously described by [42].

MTS assay

To test cell viability, Cell Titer Cell Proliferation kit according to manufacturer’s instructions were used (Promega Corporation Madison, USA). The index of viability, which is dependent on formazan generated, was evaluated using an ELISA reader, Infinite F200 (Tecan, Swiss). Test was performed in quadruplicate. The results were reported as absorbance at 492 nm.

In vitro model

The human SH-SY5Y cell line has been purchased from ECACC and cultivated in Dulbecco’s minimum essential medium, completed with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Corning, USA) at 37°C in 95% O₂ and 5% CO₂ incubator (Thermo, USA). For the PD in vitro model, the SH-SY5Y cell line was differentiated with all trans-retinoic acid (10mM) and 3 days with phorbol (80nM). At 6 DIV, cells were treated with SLAB51 0.1 mg/ml of extract for 2 hours and then added 6-OHDA (Sigma Aldrich, USA) (35μM) for 24 hours. All experiments were performed at 19^th passage and the cell culture were tested to Mycoplasma presence (Mycoplasma PCR, abm, USA).

Immunofluorescence

After culturing the cells as described above, were fixed in 4% PFA in PBS for 15 min and permeabilized in CH₃OH for 7 minutes at −20° C. Cells were incubated in 4% BSA (Sigma Aldrich, USA) for 30 minutes then with the subsequent primary overnight at 4° C: rabbit polyclonal anti-β-Tubulin III (1:1000 Abcam, Cambridge, UK), rabbit polyclonal anti-TH (1:200, Novus Biologicals, Centennial, USA), mouse monoclonal anti-GAP43 (1:200, Abcam, Cambridge, UK). After several washings, coverslips were incubated with secondary antibodies, goat anti-mouse or anti-goat IgG Alexafluor 488 or 633 or 546 (1:2000 Life Technologies, California, USA), for 1h at RT. After different washes, Vectashield mounting with DAPI (Vector Laboratories Burlingame, USA) were used. All the samples were observed using confocal laser microscope (Leica, Wetzlar, Germany).

Western blotting

Control and treated cells were collected and lysated as previously described and the protein amount were evaluated [43, 44]. 30 μg of proteins were loaded and separated on precast 4–20% gradient Bis-Tris gel in running buffer at 100 mV for 70 min followed by transfer to PVDF membranes using a semi-dry device (Thermo scientific, UK), then blocked in 5% no-fat milk for 30 minutes. Membranes were incubated with the subsequent primary antibodies overnight: anti-p-AKT (1:1000), anti-PI3K (1:1000 Cell Signaling, USA), anti p-CREB (1:500 Cell Signaling, USA), anti-pTrkB (1:2000 Cell Signaling, USA), anti-mBDNF (1:500 Abcam, UK), anti-pJNK (1:1000 Santa Cruz, USA), anti-p75(1:1000 Abcam, UK), anti-pro-BDNF(1:1000 Millipore, USA), anti-pERK5 (1:1000 Cell Signaling, USA), anti p-ERK1,2 (1:1000 Santa Cruz, USA). After different washes, membranes were incubated with 1:10000 horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein bands were detected, normalized and analyzed to actin (housekeeping). Anti-β-actin (HRP-conjugate) (1:10000) has been used. To reprobe, membranes have been stripped with Restore stripping buffer (Thermo Scientific, UK) following manufacturer’s instructions.

In vivo experiments

Animals

Animal handling and surgical procedures were performed in order to minimize discomfort and pain, according to the ethical regulations of the European Communities Council (Directive 2010/63/EU, prot #542/2019-PR).

For 6-OHDA lesion experiments, male C57BL/6 mice purchased from Charles River (Massachusetts, USA) 9-week-old were used. Animals (n=30) were kept in ventilated cages (Tecniplast, Germany) under a 12-hour light/12-hour dark cycle with water and food ad libitum. Stereotaxic (Stoelthing, USA) injections of 6-OHDA were performed as previously reported [45]. Briefly, mice were subjected to anesthesia (xylazine (10 mg/kg) and ketamine (200 mg/kg) and then 4 μg of 6-OHDA containing 0.2% L-ascorbic acid (Sigma Aldrich, USA) in saline solution or saline solution with 0.2% L-ascorbic acid (SHAM group) were injected into the right region of the striatum (coordinates relative to bregma: medial-lateral +0.18 cm; anteroposterior + 0.04 cm; dorsal-ventral +0.35 cm) with a rate of 0.5 μl/minute using single syringe nano Infusion KDS 310 (KD Scientific, USA). After injection, we waited for 5 minutes before removal.

Treatment

SLAB51 formulation was provided by Mendes Sa (Switzerland). SLAB51 was freshly prepared dissolving one sachet (1,5g/200 billion of bacteria) in 10 ml of drinking water and the treated mice received 270 μl using oral gavage (corresponding at around 5,4 billion, based on a weight human/weight mice ratio). SLAB51 was administered daily for 2 weeks previous 6-OHDA injection and followed for further 3 weeks. Control group received SLAB51 only.

Behavioral tests

Elevated body swing test

All investigators performing the behavioral tests were blinded to the treatment condition. To perform EBST, mice were gently picked up at the base of the tail and the direction of the swing, either left or right, was considered until 20 swings as described by [46].

Cylinder test

Cylinder rearing test [47] was adjusted for use in mice to evaluate forelimb use during normal exploratory behavior and was conducted before 6-OHDA lesion and 1, 2, 3 weeks after the first lesion. Each mouse was positioned in a Plexiglass cylinder 25 cm in height and 11.48 cm in diameter. Spontaneous forelimb contacts were recorded until 20 contacts for each animal (2 not consecutive times). The number of paired and impaired forelimb contacts were evaluated as percentage of total contacts observed in the entire observation time.

Morphological analysis

Animals were deeply anesthetized with ketamine/ xylazine, before being sacrificed by transcardial perfusion. Mice were perfused at RT with phosphate buffer saline (PBS), followed by 4% PFA in 0.12M phosphate buffer, pH 7.6. The brains were placed overnight in 4% PFA, then cryoprotected in 30% sucrose solution in 0.1M phosphate buffer (PB). Brains from each mouse were embedded in the OCT (Sigma Aldrich, Saint Louis, USA). The blocks were cut by a cryostat to obtain coronal 40 μm thick sections following “Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates” (Elsevier).

Immunohistochemistry

Free floating sections were incubated in a 0.3% hydrogen peroxide solution, for 10 min, protected from the light, to block internal peroxidases, and then in PBS 0.5% Triton X-100, 4% BSA for 1 h, RT. Sections were then incubated overnight at 4°C with rabbit polyclonal anti-TH (1:500), in PBS containing 0.4% Triton X-100. In control sections, the primary antibody was omitted. After incubation for 2h at RT with goat anti-rabbit IgG-HRP (Sigma, B7401), 1:100 in PBS containing 0.4% Triton X100, immuno-complexes were revealed using 3,3′-diamino-benzidine (DAB Substrate Kit for Peroxidase, Vector) as the chromogen. After extensive washing, sections were dehydrated and mounted with Permount (Fisher Scientific, US). Slides were observed with a Leica S8 Apo microscope equipped with EC3 camera. To quantify TH+ cells, 3 slices were used per each group.

Immunofluorescence

For immunofluorescence experiments, sections were processed as reported in “immunohistochemistry” section and incubated for 24 hours at 4°C with the subsequent primary antibodies: rabbit polyclonal anti-TH (1:500), anti-NeuN (1:1000), anti DAT (1:1000), anti-Iba1 (1:500), anti-GFAP (1:500). Sections were rinsed with PBS and then incubated for 2h at RT in BSA containing 0.4 % Triton X-100, and secondary antibodies Alexa488 conjugated donkey anti-rabbit IgG 1:500 or Alexa633 conjugated donkey anti-mouse IgG 1:500 and Alexa596 conjugated anti- chicken IgG 1:500 (Life Technologies, California, USA). Controls were performed by omitting the primary antibody. Image acquisition in a Leica TCS SP5 confocal microscope was performed and then analyzed by ImageJ software. In particular, the average number of Iba1 + cells per section was revealed in five regularly spaced sections of the SN per animal. To quantify GFAP intensity fluorescence, ImageJ analysis software was used and reported in the graph as fluorescence intensity/% of control, analyzing 9 different fields for each condition (n = 3 mice each group; 3 fields per mouse).

The representative mosaic images provided were subjected to rotation in order to get all the figures orientated at the same direction.

Western blotting

Under stereomicroscope, substantia nigra and striatum were isolated and the different regions were freshly lysate using pestles, protein extracted were dosed as previously described [43]. Tissues lysates containing 10μg of protein were separated on 4–13% gradient Bis-Tris gel in running buffer at 100 mV for 80 min. Proteins were transferred into PVDF membranes using a semi-dry device (Thermo scientific, UK). Membranes were washed in tris-buffered saline with 0.05% Tween20, and blocked in 5% no-fat milk for 1 h at RT. Membranes were then incubated overnight at 4°C with the following primary antibodies, diluted in the same blocking solution: anti p-NRF2 (1:5000 Abcam, UK), anti-NFKB (1:2000 Abcam, UK) anti-p-TRKB (1:2000 Cell Signaling), anti-BDNF (1:500 Abcam, UK), anti-PPARγ (1:500, Thermo, USA) anti-HO1(1:1000 Santa Cruz, USA) at 4°C overnight and then incubated with 1:10000 HRP-conjugated anti-rabbit IgG or anti-mouse IgG. Protein bands were detected with West Pico luminol (Thermo scientific) following kit’s datasheet. Through Alliance Q9 (Uvitec, Cambridge, UK) image chemiluminescent bands were detected and using ImageJ program we analyzed each band intensity normalized as indicated in the “Wester Blotting” in vitro section.

TUNEL assay

The substantia nigra (SN) region of the mouse brain was cut at 40 μm on a cryostat and stored at −80 °C. To perform terminal transferase-mediated dUTP nick end-labeling (TUNEL) analyses, sections were fixed in 4% PFA for 30minutes and then washed several times with room temperature PBS. Then, the sections were incubated in cold ethanol/acetic acid 2:1 for 5mins and washed in PBS again. The labeling of neuronal apoptosis in SN sections was performed using the apoptosis detection kit purchased from ThermoScientific (USA), which is based on the in situ TUNEL technique using terminal deoxynucleotidyl transferase (TdT) and the images though confocal microscope were acquired (Leica TCS SP5).

Statistical analysis

Statistical analysis has been performed by t-test, using PRISM 8 software. For statistical studies, *P<0.05 has been set as statistically significant.