Akkermansia muciniphila growth curve pattern and conditioned medium composition are modulated by mucin
A. muciniphila is a mucin-degrading Gram-negative bacterium of the phylum Verrucomicrobia [36]. However, the intestinal mucus layer is thought to be inversely correlated with A. muciniphila abundance in the gut [42]. Prolonged lack of dietary fibers induces damage to the mucus barrier and is directly associated with increased abundance of A. muciniphila. This would bring gut bacteria closer to the intestinal epithelium, which could trigger deleterious effects or other host compensatory responses [43]. To test whether mucin could interfere with A. muciniphila conditioned medium composition, the strain DSM-22959 was harvest and monitored for 72 hs in both BHI culture medium and BHI supplemented with 0.4% mucin (from porcine stomach, Type II) (Fig. 1a). It is clearly observed that the addition of mucin maintains the growth of A. muciniphila in BHI medium (Fig. 1a). When mucin was provided, A. muciniphila grew faster at log phase and maintained a plateau for a longer time than when cultivated in mucin-free BHI medium. In addition, when analyzing the conditioned media (CM) by MS-MS mass spectrometry, we identified 285 differentially expressed proteins in the conditioned medium obtained from A. muciniphila when cultivated for 36–40 hs (peak of growth for both conditions) in 0.4% mucin-supplemented BHI medium as opposed to 30 in mucin-free medium (Fig. 1b, Table S1). In both cases, many of these proteins have not yet been characterized by the scientific community. In summary, A. muciniphila conditioned medium is directly modulated by the presence of mucin.
Intracellular calcium signaling is elicited by Akkermansia muciniphila mucin-free conditioned medium in a model of enteroendocrine cells
Enteroendocrine cells (EECs) are chemosensory cells distributed throughout all the mucosal lining of the intestine and with their apical surface exposed to the lumen of the organ. In addition, it was recently described that EECs also connect to enteric neurons [16, 44, 45]. Due to their location at the interface between gut contents and the nervous system, EECs provide a direct route for substances in the gut to affect neural function. The STC-1 cell line is widely accepted as a model of native EECs [46] due to the expression of several gastrointestinal hormones, including cholecystokinin (CCK) and peptide YY (PYY), whose secretion pattern is compared to that of native EECs [47–49]. In addition, these cells present many neuronal-like features, including the expression of α-synuclein (αSyn) [16]. Since native EECs are hard to culture or to be collected from intestinal tissue in a sufficient number for in vitro assays, STC-1 are considered an attractive cell model for evaluating properties of EECs.
Calcium (Ca2+) is known to regulate several important cell functions, such as secretion, proliferation, apoptosis, protein biosynthesis and folding [50–52]. In order to study the effects of A. muciniphila conditioned media in the fluctuations of intracellular Ca2+ signaling in STC-1 cells, we first stimulated Fluo-4/AM-loaded cells with 1 or 10% conditioned BHI medium (BHI CM) or unconditioned BHI medium (BHI). We observed that A. muciniphila mucin-free BHI CM induces a strong increase in Ca2+ transient in a concentration-dependent manner (Fig. 2a-c). On the other hand, 0.4% mucin-supplemented BHI CM induced weaker Ca2+ signals when compared to the mucin-free condition (Fig. 2, S1a-c). In order to observe whether this Ca2+ fluctuation was due to bacterial secreted elements and not to the unconditioned culture medium, STC-1 cells were also stimulated with mucin-supplemented and mucin-free unconditioned media (BHI) and no fluctuation on intracellular Ca2+ signals was observed (Figure S2).
Therefore, secreted elements found in A.muciniphila conditioned media are key to elicit intracellular Ca2+ response in STC-1 cells.
Mucin-free A. muciniphila conditioned media increases expression of endogenous α-synuclein in STC- cells
Induced transient increase in free intracellular Ca2+ concentration by thapsigargin or Ca2+ ionophore chemical treatments lead to a significant increase in the number of cells presenting microscopically-visible αSyn aggregates [53]. Also, it is already reported that increased expression or decreased degradation of αSyn can initiate the formation of amyloid aggregates that can assemble to form Lewy bodies and Lewy neurites over the course of a lifetime [54].
In addition, misfolded αSyn is found in enteric nerves before it appears in the brain [13–15]. However, it is yet to be demonstrated whether the secreted proteins of a gut bacterium could initiate this pathologic sequence of events.
Therefore, we next analyzed whether A. muciniphila CM could modulate αSyn homeostasis in STC-1 cells. MTS assay confirmed that 48h-incubation of cells with 1 or 10% BHI CM or BHI did not decrease cell viability (Figure S3). When STC-1 cells were incubated with 1 or 10% mucin-free BHI CM for 48 hs, but not with the unconditioned one (BHI), we detected a significant clear overexpression of αSyn analyzed by immunofluorescence and Western blotting (Fig. 2d-f). However, this was not observed when the cells were incubated with 0.4% mucin-containing BHI CM (Figure S1d-f).
The SNCA gene expression in neurons, which encodes for αSyn, is known to be controlled by the GATA-2 transcription factor [55], which also plays a crucial role in central nervous system development, and erythroid cells differentiation [56]. In addition, GATA-2 has a critical role in neuronal development, particularly in cell fate specification of catecholaminergic sympathetic neurons [57, 58]. We observed that STC-1 cells not only express GATA-2 transcription factor but also exhibit increased expression of this factor when incubated with either 1% or 10% mucin-free A. muciniphila BHI CM. This supports the idea that A .muciniphila conditioned medium upregulates GATA-2 which in turn induces SNCA overexpression (Fig. 2g). To go further into the effects of A. muciniphila mucin-free conditioned media and to understand whether these phenomena were specifically due to the protein fraction of A. muciniphila conditioned medium, we stimulated STC-1 cells with 1 and 10% of heat-inactivated BHI CM. No fluctuation on Ca2+ signaling neither alteration on αSyn homeostasis evaluated by western blotting for GATA-2, αSyn and pser-129 αSyn was observed (Figure S4).
In order to confirm if these observed effects were specifically due to A. muciniphila conditioned medium, we conducted the same set of above experiments employing Escherichia coli (E. coli) conditioned medium. E. coli was chosen because it is an abundant Gram-negative microorganism from the gut. This strain was also cultivated in BHI medium under anaerobic condition as for A. muciniphila. Although we also observed a transient increase in free intracellular Ca2+ in STC-1 cells stimulated with 1 or 10% E. coli BHI CM, the amplitude of the signal was smaller than the one elicited by A. muciniphila, (Figure S5a-e). In addition, we did not detect alteration on αSyn expression levels when STC-1 cells were incubated for 48 hs with E. coli CM (Figure S5f-h).
In summary, the protein fraction of A. muciniphila mucin-free CM leads to a transient increase in free intracellular Ca2+ and induces GATA2-regulated-overexpression of αSyn in the STC-1 enteroendocrine cell model.
A. muciniphila conditioned medium induces calcium release from stores in the endoplasmic reticulum in an IP 3 -independent manner
Several maneuvers were performed to define the mechanism by which A. muciniphila mucin-free CM increases free cytoplasmic Ca2+ in STC-1 cells. To determine the source of the Ca2+, cells were stimulated in Ca2+-free medium. We observed that A .muciniphila CM induced cytoplasmic Ca2+ oscillations in a concentration-dependent manner even in Ca2+‐free medium (Fig. 3a,b). Additionally, induced-Ca2+ signals initiate/predominate in the cytoplasm (Figure S6) and were elicited in a similar fraction of STC-1 cells regardless of the presence of extracellular Ca2+. On the other hand, selective depletion of stored calcium by 10µM thapsigargin significantly blocked Ca2+ oscillations-induced by A. muciniphila CM (Fig. 3c,d). Thereby, these findings demonstrate that A. muciniphila CM sample increases cytoplasmic Ca2+ levels by mobilizing intracellular Ca2+ stores.
A classic manner by which extracellular factors initiate an intracellular Ca2+ mobilization is by generating InsP3 to bind and release Ca2+ from InsP3 receptors in the endoplasmic reticulum [59]. In order to investigate whether the cytoplasmic Ca2+ increase was triggered by InsP3 generation, we stimulated STC-1 cells in the presence of the InsP3 receptor inhibitor xestospongin C [60]. Incubation of cells for 30 min and continuous perfusion with 2.5 µM xestospongin C did not impair A. muciniphila CM -induced Ca2+ mobilization, suggesting an InsP3-independent release of intracellular Ca2+ stores (Fig. 3e,f). To go further into the mechanism by how A. muciniphila CM evokes Ca2+ release from intracellular stores, we incubated cells for 30 min with dantrolene (75 µM), an inhibitor of Ca2+ release through ryanodine receptor (RYR) channels [61–63]. In the presence of 75 µM dantrolene, only a very small Ca2+ increase was observed following stimulation with 1 or 10% A. muciniphila CM (Fig. 3g,h).
Thus, dantrolene eliminated A. muciniphila CM Ca2+ response in enteroendocrine cells. Taken together, these results show that proteins contained in A. muciniphila conditioned medium works as a physiological RYR gating agent, eliciting intracellular Ca2+ signals by directly modulating RYR in the cytoplasm.
Mitochondrial calcium overload and impaired membrane potential (∆Ψm) is elicited by A. muciniphila conditioned medium
Global changes in Ca2+ homeostasis accompanied by the alteration in cellular bioenergetics status and thereby imposing oxidative stress in cells are reported in PD [64, 65]. The cytosolic Ca2+ concentration in unstimulated cells is maintained at low levels (∼100 nM) by several enzymes that translocate Ca2+ ions into intracellular stores or across the plasma membrane. Moreover, Ca2+ uptake into the mitochondria is not limited to the control of organelle function, but also has a direct impact on the intracellular Ca2+ signals evoked by agonist stimulation in the cytosol through modulation of their kinetics and spatial dimensions [66]. Enhanced cytosolic Ca2+ concentration, on the other hand, affects the bioenergetics of the cells by promoting increased ATP demand [67]. Furthermore, this alteration in cytosolic Ca2+ hampers the normal Ca2+ handling by various intracellular organelles, including mitochondria, and threatens neuronal survival. Although well established for neuronal cells, there is still a gap regarding changes in mitochondrial Ca2+ dynamics in enteroendocrine cells due to gut microbiome stimulation and how this event might be related to αSyn homeostasis.
Thereby, we aimed at evaluating mitochondrial Ca2+ under stimulation with A. muciniphila conditioned medium. When STC-1 cells loaded with the mitochondrial Ca2+ indicator Rhod-2/AM dye were stimulated with 10% of A. muciniphila CM, we observed a significant increase in mitochondrial Ca2+ uptake when compared to unconditioned BHI medium (Fig. 4a,b). In addition, when we incubated the cells for 48 hs with 1 or 10% CM and stimulated with ATP (10 µM), mitochondrial fluorescence was dramatically increased in the group incubated with 10% BHI CM when compared to 1% BHI CM or unconditioned BHI medium (1 and 10%) suggesting that long exposure to A. muciniphila conditioned medium induces increased uptake of Ca2+ by the mitochondria (Fig. 4c,d).
As previously mentioned, enhanced, or sustained Ca2+ stress results in mitochondrial injury due to Ca2+ overload. Excessive mitochondrial Ca2+ uptake or impaired Ca2+ efflux influences mitochondrial membrane potential (∆Ψm) leading to depolarization of mitochondrial inner membrane, swelling of the organelle, and ultimately cell death [68–70]. In order to observe whether mitochondrial Ca2+ uptake induced by A. muciniphila CM could lead to mitochondrial damage, we monitored ∆Ψm in STC-1 cells under A. muciniphila CM incubation for 48 hs. After treatment, cells were stained with the mitochondrial-targeted probe Mitotracker Red CMXRos, which accumulates in mitochondria depending on its membrane potential and has been widely used as an indicator of reduced ∆Ψm [71, 72]. As can be observed on Fig. 4e,f, cells incubated with 10% CM presented a reduced fluorescent signal of the probe what suggests impaired membrane potential.
Altogether, the results described so far demonstrate that A. muciniphila mucin-free conditioned medium induces exacerbated mitochondrial Ca2+ uptake, which in turn is the driven force that causes mitochondrial damage, reflected by a loss of membrane ∆Ψm.
Increased intracellular ROS level, α-synuclein phosphorylation and aggregation as a consequent event of A. muciniphila conditioned medium stimulation of enteroendocrine cells
It is suggested that endogenous ROS mainly modulate cell signaling locally and stimuli that promote ROS formation or mitochondrial alterations highly correlate with mutant αSyn phosphorylation at Serine 129 (Ser129), a promoter of αSyn aggregation propensity and toxicity in PD [73–75]. Therefore, we next measured intracellular levels of ROS under stimulation with 1 or 10% A. muciniphila CM by live cell imaging. STC-1 cells were incubated for 30 min with DHE and continuously perfused with buffer containing 1 or 10% CM. Buffer/unconditioned media and H2O2 (100 µM) perfusion were used as negative and positive controls, respectively. The real-time fluorescence measurement indicates that the surge of ROS level after H2O2 or 1–10% CM stimulation was significantly higher than stimulation with buffer or 1–10% unconditioned BHI media for 5 min (Fig. 5a,b). In addition, cells stimulated with 1 or 10% CM presented increased DHE fluorescence in a similar manner.
As mentioned, stimuli that promote intracellular ROS formation and mitochondrial damage highly correlate with αSyn phosphorylation at Ser129, an event that may precede cell degeneration in PD [73]. Previous observations have shown that both nigral and dorsal motor nucleus of the vagus nerve neurons present a high vulnerability to oxidative challenges [76]. Since the nigro-vagal pathway that controls gastric tone and motility connect these brain regions, it raises the possibility that an oxidative injury may be relayed and possibly amplified through this anatomical and functional connection.
In order to evaluate whether increases ROS levels induced by A. muciniphila CM could promote αSyn phosphorylation and aggregation, we incubated the cells for 48 hs in the presence of CM or unconditioned BHI media and directed them to immunofluorescence and Western blotting. Confocal microscopy images showed strong deposits of pSer129-αSyn in STC-1 cells incubated with 1 and 10% CM (Fig. 5c). In addition, quantification by Western blotting showed a 2-3-fold increase of p-Ser129-αSyn in cells treated with the conditioned medium when normalized against total αSyn (Fig. 5d). To establish whether A. muciniphila CM-induced p-Ser129 αSyn might play a role on αSyn aggregation in our STC-1 cell model, we transfected cells with full-length human αSyn-GFP-tagged and incubated them with unconditioned or CM for 48hs. Unconditioned BHI media (1 or 10%) did not cause αSyn to form cellular inclusions. However, 1 and 10% CM led to the formation of small to large αSyn granules within the cytoplasm (Fig. 5e). When we quantified the number of GFP-positive cells containing intracellular aggregates, we observed that over 50% of the cells stimulated with A. muciniphila CM contained αSyn granules (Fig. 5f). Thereby, conditioned medium of A. muciniphila grown in the absence of mucin induces intracellular αSyn aggregation in enteroendocrine cell model.
Oral administration of Akkermansia muciniphila to aged mice leads to αSyn aggregation in CCK-positive enteroendocrine cells
So far, our results showed that the protein fraction of A.muciniphila conditioned medium grown in the absence of mucin induces mitochondrial stress and ROS generation which in turn led to αSyn aggregation. In addition, previous works have shown that aged mice have impaired mucus barrier in the colon and ileum and this thinner mucus layer was associated with increased bacterial penetrability and contact with the epithelium[42, 77]. Therefore, we wondered whether the increased levels of A.muciniphila in aged mice could be a trigger to αSyn pathology in the gut. To assess if A.muciniphila could cause motor deficits, we treated aged mice with bacterial cells (AKK group) for 28 continuous days (Figure S7a). After 28 days of oral administration, AKK group did not exhibit alteration in body weight but presented significantly higher number of A. muciniphila 16S rRNA copies in stool (Figure S7b,c).
We used three measures of gross motor function: time to cross a challenging beam, the cylinder test and wire hanging. In none of the test we observed differences between control and AKK group (Figure S7d-f). Utilizing an antibody that recognizes only conformation-specific αSyn aggregates and fibrils, we performed dot blot analysis for aggregated αSyn in total protein extract from ileum of control and A.muciniphila-treated animals and observe similarly low levels of αSyn aggregation in both groups (Fig. 6a,b). Interestingly, by immunofluorescence, we observe αSyn aggregation in cholecystokinin (CCK) -positive enteroendocrine cells (Fig. ¨6c,d). In addition, the number of CCK-positive cells containing αSyn aggregates in AKK group was ~ 4 times higher when compared to control animals which barely presented αSyn-aggregate-containing cells (Fig. 6e). These data suggest that A.muciniphila, when exposed to a mucin-deprived environment, regulates pathways that promote αSyn aggregation and/or prevent the clearance of insoluble protein aggregates in enteroendocrine cells, suggesting that αSyn pathology can indeed start in the gut.
Mitochondrial calcium buffering reverts the damaging effects to mitochondria and prevents α-synuclein aggregation
Inhibition of mitochondrial Ca2+ uptake was shown to diminish the oxidative stress in substantia nigra pars compacta dopaminergic neurons (SNpc DNs) suggesting that mitochondrial oxidative stress could also be due to mitochondrial Ca2+ overload [78]. Several lines of investigation point out to mitochondrial Ca2+ imbalance as key factor to be modulated in order to control the progression of PD. In order to observe whether modulating mitochondrial Ca2+ in enteroendocrine cells could reverse intracellular ROS generation and αSyn aggregation, we transfected the cells with parvalbumin (PV) fused to a mitochondrial targeting sequence (MTS) and GFP [41]. Parvalbumin (PV) is a cytosolic Ca2+-binding protein of the large EF-hand protein family, involved in intracellular Ca2+ regulation and buffering. GFP targeted to the mitochondrial matrix was used as a control (MTS-GFP) (Fig. 7a).
One or 10% BHI CM elicited a robust increase in mitochondrial Ca2+ in cells expressing MTS-GFP alone, but this was reduced by approximately 90% in cells expressing PV in mitochondria (Fig. 7b,c). These results demonstrated that PV-MTS‐GFP was correctly targeted to the mitochondrial matrix and efficiently buffered mitochondrial Ca2+ overload driven by stimulation with A. muciniphila conditioned medium.
Once mitochondrial Ca2+ was buffered, the next set of experiments aimed to observe whether the damaging effects caused by A. muciniphila conditioned medium could be prevented. When we stimulated the cells expressing PV-MTS construct with 1 and 10% BHI CM, the increase in intracellular ROS was significantly suppressed (Fig. 7d,e) indicating that mitochondrial Ca2+ buffering prevents intracellular oxidative stress.
To test the effect of mitochondrial Ca2+ on Ser129-phosphorylation of αSyn induced by A. muciniphila conditioned medium, we incubated the transfected cells with 1 or 10% CM for 48hrs. Total cell lysate evaluated by Western blotting showed that levels of Ser129-phosphorylated αSyn significantly decreased in PV-MTS expressing cells when compared with control cells (MTS-GFP) (Fig. 8a,b). However, no effect was observed in the total expression level of αSyn, which remained higher when compared to untreated cells (Fig. 8a,c).
We then extended our observation that mitochondrial Ca2+ can suppress intracellular ROS generation and αSyn phosphorylation to the formation of αSyn aggregates. Hence, we double-transfected cells with the PV-MTS-GFP construct and human αSyn mCherry-tagged. Large number of αSyn aggregates were observed in cells expressing the control construct (MTS-GFP) after 48 hs of treatment with 1 or 10% conditioned medium. However, the number of αSyn aggregates in cells expressing the PV-MTS-GFP constructed was markedly reduced (Fig. 8d,e).
Taking together, these findings provide evidence on the mechanism by which A. muciniphila conditioned media induces αSyn aggregation in enteroendocrine cells (Fig. 9).