urdA-coding microbial enzyme, responsible for producing imidazole propionate, is enriched in the gut microbiome of patients with PD

To identify species with differential abundances in the gut microbiome of individuals with PD, we conducted an unbiased metagenome-wide association study by reanalyzing publicly available dataset. Our analysis confirmed Bifidobacterium dentium and S. mutans as the most significantly associated species with PD, both showing increased abundance in PD (Supplementary Fig. 1a and Fig. 1a). While B. dentium was notably enriched, it is frequently detected in the feces of healthy individuals and has been implicated in beneficial roles, such as reducing colonic inflammation, improving the intestinal mucus layer, and normalizing repetitive and anxiety-like behaviors through serotonergic regulation. Given these potentially protective roles, we focused our investigation on S. mutans to explore its potential pathogenic role in PD. Specifically, we investigated whether the level of urdA (gene encoding urocanate reductase, an enzyme for imidazole propionate production) is altered in the gut microbiome of individuals with PD. Thus, we analyzed the abundance of urdA and hutH (encoding histidine ammonia lyase responsible for converting histidine to urocanate) using published whole-genome shotgun sequencing datasets of fecal samples from 491 individuals with PD and 234 neurologically healthy elderly controls. Our analysis showed significantly higher levels of urdA in patients with PD than in neurologically healthy controls, whereas hutH levels showed no significant differences (Fig. 1b, c). Furthermore, urdA from S. mutans, but not from other Streptococcus species, was significantly more prevalent in PD patients (Supplementary Fig. 1b, c), supporting the rationale for investigating the potential role of the UrdA-possessing bacterial strain S. mutans and imidazole propionate in PD pathology.

To investigate whether S. mutans colonization in the gut contributes to PD pathology, germ-free (GF) C57BL6/N mice were colonized with S. mutans by administering 10 CFU (Colony-Forming Unit) per mouse via gavage once weekly for 28 days (totaling three gavages) (Fig. 1d). To assess the importance of S. mutans's metabolic activity, pasteurized S. mutans were administered in the same manner (Fig. 1d). The 28-day S. mutans colonization of GF mice did not affect body weight, cecum weight, or brain weight compared to the vehicle or pasteurized S. mutans-gavaged groups (Supplementary Fig. 1d-f). Successful colonization and survival of S. mutans within the GF mouse gut was confirmed by viable colonies in feces (Supplementary Fig. 1g and Fig. 1e). Additionally, absolute quantification of the V3-V4 regions of the 16S rRNA gene and the S. mutans 16S gene revealed significantly higher S. mutans copy numbers in the S. mutans-gavaged group compared to the pasteurized S. mutans-gavaged group (Supplementary Fig. 1h), suggesting that S. mutans not only colonizes the mouse gut but also thrives in it. The colonization of S. mutans was further examined throughout the length of the intestine (Supplementary Fig. 1i, j) and showed robust colonization preferentially in the distal gut, particularly the ileum and colon, of S. mutans-gavaged GF mice.

PD is defined as the selective loss of dopaminergic neurons. Therefore, we sought to determine whether gut-colonized S. mutans induces neurotoxicity in the midbrain of GF mice. S. mutans monocolonization in GF mice was sufficient to reduce the number of tyrosine hydroxylase (TH)-positive and Nissl-stained dopaminergic neurons in the substantia nigra pars compacta, as well as TH-positive dopaminergic processes in the substantia nigra reticularis, indicating dopaminergic neurodegeneration in the midbrain (Fig. 1f). In contrast, pasteurized S. mutans colonization failed to induce dopamine-cell death (Fig. 1f), supporting the fact that S. mutans must be metabolically active to exert its pathogenicity. Consistent with this, the density of TH-positive dopaminergic axon terminals in the striatum was also reduced in a manner dependent on S. mutans metabolic activity (Supplementary Fig. 1k). However, Nissl-stained neuronal counts in the substantia nigra reticularis (Fig. 1f) and the prefrontal cortex were similar among the three experimental groups (Supplementary Fig. 1l), suggesting that S. mutans-induced neurotoxicity is brain region-specific. Moreover, S. mutans induced reactive astrogliosis in the ventral midbrain but not in the cortex, as demonstrated by a more than 3-fold increase in GFAP staining in the substantia nigra pars compacta compared to that in vehicle-treated controls (Fig. 1g, and Supplementary Fig. 1m). In contrast, metabolically inactive pasteurized S. mutans-treated mice did not show astrogliosis in the ventral midbrain (Fig. 1g). S. mutans also induced microgliosis, as indicated by increased Iba1 staining and enlarged microglial soma size, which are established markers of microglial activation (Fig. 1h). Reflecting these brain pathologies, motor deficits were induced in GF mice colonized with S. mutans, but not in those colonized with pasteurized S. mutans. as assessed by the pole test showing a significantly delayed latency to complete the pole (Fig. 1i). This supports the hypothesis that the metabolic activity of this bacterium plays a critical role. Our results indicate that the PD-associated increase in S. mutans in the gut can induce PD-related changes, such as selective loss of dopaminergic neurons, astrogliosis, microgliosis, and motor deficits.

PD-associated S. mutans harbors urdA, which is more abundant in the gut microbiome of patients with PD (Fig. 1b), while its activity is responsible for imidazole propionate production. Thus, we investigated whether gut-colonized S. mutans can produce imidazole propionate in vivo and whether it can enter the circulation, reaching the brain tissue. We observed that the levels of imidazole propionate produced by gut-colonized S. mutans, but not by pasteurized S. mutans, increased in the blood, reaching the brain tissue (Fig. 1j, k), while the concentration of its precursor urocanate was not affected (Supplementary Fig. 1n, o). Taken together, we showed that gut-colonized, metabolically active S. mutans in GF mice produced imidazole propionate, resulting in a marked increase in blood imidazole propionate levels and its penetration into the brain. These results suggested that the PD-associated increase in S. mutans in the human gut may contribute to the elevation of blood and brain imidazole propionate concentrations.

Given that urdA from S. mutans was significantly more prevalent in PD patients (Supplementary Fig. 1b) and that S. mutans may exert pathogenic effects through additional mechanisms, we heterologously expressed urdA from S. mutans in an Escherichia coli MG1655 strain lacking urdA. While E. coli MG1655 did not produce imidazole propionate, E. coli expressing S. mutans urdA produced imidazole propionate at levels comparable to those produced by S. mutans (Fig. 2a). Importantly, urdA expression did not affect the growth rate of E. coli (Supplementary Fig. 2a). Colonization levels of both E. coli strains in the GF mouse gut were comparable (Fig. 2b, c, Supplementary Fig. 2b). No significant differences were observed in body weight, cecum weight, or brain weight among the groups (Supplementary Fig. 2c, d, e). Although S. mutans colonized both the distal small intestine and cecum, E. coli primarily colonized the cecum (Supplementary Fig. 2f), yet gut-colonized E. coli expressing urdA effectively produced systemic imidazole propionate, reaching the brain (Fig. 2d, e) without altering urocanate levels (Supplementary Fig. 2g, h). Similar to S. mutans, gut-colonized E. coli expressing urdA induced dopaminergic neurodegeneration (Fig. 2f), astrogliosis, microgliosis (Fig. 2g, h), and motor dysfunction (Fig. 2i). Consistent with dopaminergic neurodegeneration, mice colonized with urdA-expressing E. coli showed a marked reduction in dopaminergic axon terminal density in the striatum, as well as diminished dopaminergic processes in the substantia nigra reticularis (Supplementary Fig. 2i, Fig. 2f). However, neuronal numbers in the substantia nigra reticularis (Fig. 2f) and GFAP expression in the cortex remained unaffected (Supplementary Fig. 2j), again demonstrating region-specificity. Taken together, these findings identify UrdA as a key pathological factor that drives PD-related neurodegeneration, astrogliosis, microgliosis, and motor dysfunction, reinforcing the potential role of gut microbiota in PD pathology.

Next, we sought to determine the molecular mechanisms underlying dopaminergic toxicity induced by S. mutans. Imidazole propionate altered insulin signaling by activating the p38γ-mTORC1 pathway in hepatocytes. The activation of the mTORC1 pathway has been implicated in aging and neurodegenerative diseases. Imidazole propionate produced by gut-colonized S. mutans reached the brain (Fig. 1k), therefore, we investigated whether this colonization is sufficient to induce mTORC1 activation in the midbrain. Intriguingly, gut-colonized S. mutans in GF mice, and not pasteurized S. mutans, led to phosphorylation of S6 and 4E-BP1, surrogate markers of mTORC1 activation, specifically in dopaminergic neurons (TH-positive) of the substantia nigra pars compacta, without affecting mTORC1 signaling in non-dopaminergic neurons in the substantia nigra reticularis (Fig. 3a, and Supplementary Fig. 3a) or the prefrontal cortex (Supplementary Fig. 3b). Similarly, gut-colonized E. coli expressing urdA increased S6 phosphorylation in TH-positive dopaminergic neurons of the substantia nigra pars compacta but not in the substantia nigra reticularis (Fig. 3b). This relatively selective mTORC1 activation in dopaminergic neurons was consistent with their observed loss of dopaminergic neurons in the ventral midbrain without involving cortical neuron pathology in GF mice colonized with S. mutans (Fig. 1f). While imidazole propionate exerted relative regional specificity in inducing brain pathology in vivo, mTORC1 activation was still elicited in primary cortical neurons following imidazole propionate treatment in vitro, as evidenced by the phosphorylation of S6K1 at T389 and subsequent downstream signaling events, such as serine phosphorylation of IRS1 (Fig. 3c). These effects were effectively blocked by the mTORC1 inhibitor rapamycin or p38γ inhibitor pirfenidone (Fig. 3c), in line with the previous findings in primary hepatocytes. Additionally, imidazole propionate treatment of primary cultured cortical neurons resulted in ~50.3% neurotoxicity, which was effectively blocked by both rapamycin and pirfenidone treatments (Supplementary Fig. 3c). These results underscored the key role of the imidazole propionate/p38γ/mTORC1 pathway in imidazole propionate-induced neuronal toxicity.

To investigate whether mTORC1 inhibition can reverse gut-colonized S. mutans-induced PD pathology, we depleted the gut microbiome of C57BL/6 N mice by administering an antibiotic cocktail (Abx) via oral gavage twice daily for seven days (Fig. 3d). This method, as described previously, effectively reduces the bacterial load (Supplementary Fig. 3d), as monitored by a colony formation assay with fecal samples. Starting on the 8 day, the antibiotic-treated mice were gavaged with S. mutans (10 CFU/mouse) daily for 14 days with or without an intraperitoneal injection of rapamycin (Fig. 3d). S. mutans was undetectable before (Pre-Abx) and seven days after antibiotic treatment (Post-Abx), but its levels markedly increased following colonization. Rapamycin did not affect S. mutans colonization efficiency, as demonstrated by absolute quantification of the S. mutans 16S rRNA gene (Supplementary Fig. 3e). These treatments did not affect body weight or cecal weight (Supplementary Fig. 3f, g). Interestingly, S. mutans-induced decrease in brain weight in the antibiotic-treated mice was reversed by rapamycin treatment (Supplementary Fig. 3h). Although antibiotic-treated mice exhibited higher plasma and brain imidazole propionate levels than GF mice (Figs. 1j, k and 3e, f), S. mutans colonization further increased these levels to ~400 nM (Fig. 3e). Similarly, brain imidazole propionate concentration increased robustly in antibiotic-treated mice colonized with S. mutans (Fig. 3f). Importantly, rapamycin treatment did not reduce the elevated levels of imidazole propionate induced by S. mutans in plasma and brain (Fig. 3e, f). Precursor urocanate levels were similar among all experimental groups and were not affected by S. mutans colonization or rapamycin treatment (Supplementary Fig. 3i, j). The increase in S6 and 4E-BP1 phosphorylation induced by S. mutans in the dopaminergic neurons of the substantia nigra was almost completely reversed by rapamycin treatment (Fig. 3g, Supplementary Fig. 3k), despite comparable elevations in imidazole propionate in the brain of S. mutans-colonized mice with or without rapamycin (Fig. 3f). In support of our hypothesis of mTORC1-dependent neurotoxicity, rapamycin treatment (mTORC1 inhibition) effectively prevented 4E-BP1 phosphorylation, dopaminergic neurodegeneration, astrogliosis, and microgliosis in the ventral midbrain, as well as the loss of dopaminergic processes in the substantia nigra reticularis and dopaminergic axon terminals in the striatum (Fig. 3h, i, and Supplementary Fig. 3k, l, m). Rapamycin also reversed motor dysfunction induced by S. mutans colonization in antibiotic-treated mice (Fig. 3j).

PD is characterized by aberrant α-Syn aggregation in Lewy bodies and Lewy neurites, and mTORC1 activation is implicated in α-Syn aggregation, therefore, we investigated the potential interaction between imidazole propionate and α-Syn aggregation pathologies. We employed well-established PD neuron models in which α-Syn aggregation and neurotoxicity can be efficiently induced by introducing in vitro prepared α-Syn preformed fibrils (PFF). PFF was prepared as previously described and their amyloid conformation and aggregate formation were confirmed by Western blot and thioflavin T (ThT) fluorescence assays (Supplementary Fig 4a-c). Subsequently, PFF was sonicated into smaller fragments, and the pathogenic capacity of these sonicated PFF fragments was validated by the induction of pSer129-α-Syn-positive aggregates in primary cortical neurons (Supplementary Fig. 4d). Treatment of primary cortical neurons with a low dose of 0.1 μg/mL α-Syn PFF for seven days resulted in a trend of Lewy-like inclusions and Lewy neurites formation, enriched in pS129-α-Syn (Fig. 4a, b). Treatment with imidazole propionate potentiated the low-dose PFF-induced Lewy inclusion formation (Fig. 4a, b). Co-treatment with 1 μM imidazole propionate resulted in a significant elevation of pS129-α-Syn positive inclusions compared with PFF treatment alone (Fig. 4a, b). Additionally, nanomolar concentrations of imidazole propionate markedly increased neuronal toxicity, as evidenced by PFF-induced neurite fragmentation observed through MAP2 (neuronal markers) immunofluorescence (Fig. 4a, c). To quantitatively assess exacerbated α-Syn aggregation, we analyzed α-Syn levels in Triton X-100-soluble and -insoluble protein fractions by Western blotting. Treatment with 10 nM imidazole propionate in cortical neurons exposed to low-dose PFF led to a marked accumulation of α-Syn aggregates in the Triton X-100-insoluble fraction, indicating a pathological interaction between imidazole propionate and α-Syn seeding (Supplementary Fig. 4e).

Therefore, we investigated whether gut-colonized S. mutans exacerbates PFF-induced α-synucleinopathy in vivo. After depleting the microbiome with antibiotics for seven days, 10 µg of PFF was stereotaxically injected into the substantia nigra to establish a sporadic PD mouse model with α-synucleinopathy. Four h later, the mice were colonized with S. mutans daily for 21 days (Fig. 4d). Gut colonization by S. mutans significantly exacerbated the aggregation of insoluble α-Syn induced by nigral PFF injection (Supplementary Fig. 4f). In antibiotic-treated mice, PFF injection alone led to mTORC1 activation in TH-positive dopaminergic neurons in the substantia nigra (Fig. 4e, Supplementary Fig. 4g). This activation was further increased by S. mutans colonization (Fig. 4e, and Supplementary Fig. 4g). Similarly, there was a dose-dependent activation of mTORC1 by imidazole propionate in primary cortical neurons seeded with α-Syn PFF, indicated by a gradual increase in S6K1 phosphorylation (Supplementary Fig. 4h). Furthermore, nigral PFF injection into antibiotic-treated mice led to modest formation of Lewy-like inclusions, which were markedly enhanced by S. mutans colonization (Fig. 4f). In line with these findings, PFF-induced dopaminergic neurodegeneration (Fig. 4g), loss of dopaminergic fibers (Fig. 4g, and Supplementary Fig. 4i), astrogliosis, microgliosis (Supplementary Fig. 4j, k), and motor deficits [assessed by the pole test (Fig. 4h) and rotarod test (Fig. 4i)] in antibiotic-treated mice were significantly aggravated by S. mutans colonization. This rapid onset of PFF-induced PD is unusual compared to the typical ~3 months incubation period required for dopamine cell loss in wild-type mice. This rapid progression might be associated with gut sterilization by antibiotics, as the same PFF dose and treatment duration in non-antibiotic-treated mice did not result in dopaminergic neuron loss, despite a modest increase in pS129-α-Syn levels (Supplementary Fig. 4l, m). Taken together, these findings suggest that gut-colonized S. mutans and potentially its metabolite imidazole propionate can worsen pre-existing α-synucleinopathy and its related pathologies in PD via mTORC1 activation.

Given that imidazole propionate is not the sole metabolite produced by S. mutans, we sought to determine whether imidazole propionate alone induces PD pathology in vivo. To evaluate its ability to cross the blood-brain barrier and distribute across different brain regions, we first administered imidazole propionate systemically via intraperitoneal injection of either vehicle or imidazole propionate for three days, a dosage shown to induce nanomolar blood concentrations. This treatment increased imidazole propionate levels to approximately three pmole/mg in the brain (Supplementary Fig. 5a), without affecting the concentration of its precursor urocanate (Supplementary Fig. 5b), therefore, confirming the penetrability of this metabolite through the blood-brain barrier.

Consistent with the observations in S. mutans-colonized mice, systemic administration of imidazole propionate for 21 days led to mTORC1 activation (Fig. 5a, and Supplementary Fig. 5c), TH-positive dopaminergic neuron reduction in the substantia nigra pars compacta (Fig. 5b), motor dysfunction (Fig. 5c, d), and astrogliosis (Supplementary Fig. 5d). Additionally, the density of TH-positive dopaminergic axon terminals decreased in the striatum of mice administered with systemic imidazole propionate compared to those in the vehicle control (Supplementary Fig. 5e). However, the Nissl-stained neuronal counts in several hippocampal subregions, including the dentate gyrus, CA1, and CA3, were similar between the control and imidazole propionate-treated groups (Supplementary Fig. 5f), consistent with the lack of impairment in cognitive function and spontaneous exploratory activities assessed by the Barnes maze and open field tests, respectively (Supplementary Fig. 5g, h). Although imidazole propionate efficiently penetrated the hippocampus (Supplementary Fig. 5a), it did not cause neuronal death in this region (Supplementary Fig. 5f). This suggests that brain pathologies induced by imidazole propionate are specific to midbrain dopaminergic neuron, although further studies are warranted to evaluate potential long-term effects.

To further confirm the direct pathogenic effects and molecular mechanism of imidazole propionate on midbrain dopamine neurons, we stereotaxically injected this metabolite into the substantia nigra of mice brains (Fig. 5e). Three days post-injection, we observed mTORC1 activation (Fig. 5f, and Supplementary Fig. 5i), and robust dopaminergic neurodegeneration in the substantia nigra pars compacta and striatum of mice injected with imidazole propionate (Fig. 5g, Supplementary Fig. 5j). This imidazole propionate-induced neurotoxicity appeared to selectively affect dopamine neurons (Fig. 5g), as Nissl-stained neurons in the substantia nigra reticularis remained unaffected (Fig. 5g). Along with mTORC1 activation and dopamine neuron loss, acute brain injection of imidazole propionate resulted in marked astrogliosis and microgliosis (Fig. 5h, and Supplementary Fig. 5k) and severe motor deficits (Fig. 5i, j), similar to those induced by gut-colonized S. mutans or systemic imidazole propionate administration. These brain-injected imidazole propionate-induced pathologies were partially inhibited by rapamycin treatment (Fig. 5f-j). Taken together, our findings suggest a pathological role for gut-derived microbial imidazole propionate in selective dopaminergic neuronal loss in PD, partly mediated by the mTORC1 pathway.

Our observations indicated that microbial imidazole propionate can represent a contributing factor to PD development. To further explore this, we measured imidazole propionate concentrations in the plasma of 65 individuals with PD (mean disease duration: 9.5 years) and 65 age- and sex-matched neurologically healthy participants (Supplementary Table 1). Individuals with type 2 diabetes exhibited elevated imidazole propionate levels compared with those with normal glucose tolerance. However, in our cohort, only six patients had type 2 diabetes (Supplementary Table 1), minimizing the potential contribution of this condition to imidazole propionate levels in patients with PD. Our results revealed significantly higher levels of imidazole propionate in individuals with PD than those in neurologically healthy, age-matched controls (Fig. 5k). These findings suggested a potential association between microbial imidazole propionate and PD development in humans.