This is the first study to investigate the possible role of gut microbial communities in Tuberous Sclerosis Complex (TSC), with a specific focus on epilepsy

Tuberous sclerosis (TSC) is a rare genetic, autosomal-dominant, multisystem disease (Crino et al., 2006), with an incidence of approximately 1 case per 6,000–10,000 live births (Henske et al., 2016).

TSC is caused by mutations in the TSC1 or TSC2 genes encoding for hamartin and tuberin, respectively. These proteins form a complex that regulates the mammalian/mechanistic target of rapamycin (mTOR), and the hyperactivation of mTOR, caused by loss of TSC1 or TSC2, is involved in the formation of benign tumors. Therefore, patients with TSC (pwTSC; Laplante and Sabatini, 2012) may develop benign tumors in several organs, including kidneys (Neumann et al., 1998), lungs (Vicente et al., 2004), heart (Jóźwiak et al., 2005), and central nervous system (CNS; Gomez et al., 1999). Lesions in the CNS, such as cortical tubers and subependymal astrocytomas, may cause intellectual disability (ID) and behavioral disorders. Indeed, most individuals exhibit tuberous-sclerosis-associated neuropsychiatric disorders (TANDs) during their lives, and children have an increased risk of developing Autism Spectrum Disorder (ASD; Sparagana and Roach, 2000). TSC is associated with epilepsy in 70–90% of patients, frequently manifesting with medication-resistant seizures (White et al., 2001; Chu-shore et al., 2010; Curatolo et al., 2015; Vignoli et al., 2013). While seizures are generally thought to originate from cortical tubers, marked by dysmorphic neurons and giant cells, the exact mechanism of epileptogenesis in TSC remains complex and not fully understood (Rastin et al., 2023).

The microbiota-gut–brain axis (MGBA) has recently been widely investigated in the etiopathogenesis of epilepsy, both in animal models and human studies (Zhang et al., 2025; da Silva et al., 2025; Ceccarani et al., 2021; Riva et al., 2025). Intestinal microorganisms may contribute to seizure onset and medication resistance through various mechanisms, including the promotion of a basal inflammatory state (Zhao et al., 2023), altering gastrointestinal barrier homeostasis, and the production of a wide range of bioactive metabolites (Wells et al., 2017). Gut bacteria produce both neuroactive compounds, such as tryptophan, serotonin, and dopamine, which can reach the central nervous system through the bloodstream or influence neurons in the enteric nervous system (Stilling et al., 2014) and other relevant metabolites such as short-chain fatty acids (SCFAs; Wells et al., 2017). These latter, particularly butyric acid, exert diverse effects that may be relevant to epilepsy, including epigenetic modulation, neuroprotection, and both local and systemic anti-inflammatory actions (Kalkan et al., 2025). By influencing neuronal excitability, synaptic plasticity, and inflammatory pathways, SCFAs could contribute to seizure mitigation and improved neurological outcomes (Yan et al., 2025).

Since pathogenic variants alone cannot account for the broad spectrum of clinical manifestations in pwTSC, in this pilot study, we investigated whether the gut microbiota might contribute to the severity of comorbidities, particularly seizure occurrence, through the gut-brain axis.

To our knowledge, this is the first study investigating the possible contribution of the gut microbial communities to the neurological features of TSC, especially regarding epilepsy.

In recent years, the gut microbiota has garnered increasing interest in the field of neuroscience, although its role although its role in epilepsy is still in its early stages, several promising findings have already emerged (Ceccarani et al., 2021; Riva et al., 2025; Zhu et al., 2024). Indeed, alterations in microbiota composition have been reported in individuals with epilepsy as well as in certain animal models (Zhu et al., 2024). Both preclinical and clinical studies suggest that modulating the gut microbiota may have antiseizure effects, highlighting its potential not only as a biomarker but also as a therapeutic target (Gong et al., 2021; He et al., 2017; Gómez-Eguílaz et al., 2018). For instance, recent studies demonstrated the beneficial effect of probiotics as an adjunctive treatment in drug-resistant epilepsy (Gómez-Eguílaz et al., 2018; El-Sharkawy et al., 2024).

TSC is associated with epilepsy in 70–90% (Curatolo et al., 2015; Vignoli et al., 2013) and can cause developmental epileptic encephalopathy due to early onset epilepsy and associated neurodevelopmental disorders (Jóźwiak et al., 2025) and undoubtedly linked to the genetic substrate underlying the disorder (Ng et al., 2022). Generally, patients carrying TSC2 pathogenic variants, as the majority of the subjects enrolled in the present study, present a more severe phenotype, characterized by a higher number of tubers, earlier age at seizure onset, and higher prevalence of ID (Curatolo et al., 2023). Nevertheless, the clinical phenotype may show a high variability, and recent preclinical studies and human reports have suggested a possible role of inflammatory processes, particularly the activation of microglia, increased expression of pro-inflammatory cytokines, as well as aberrant mTOR-mediated immune responses, in the development and progression of neurological symptoms in pwTSC (Kaur et al., 2021; Xie et al., 2020; Fuso et al., 2016; Balthazard et al., 2025; Gruber et al., 2022).

A recent study in a Tsc2^+/− mouse model showed that deficiency of the TSC2 gene causes different gut microenvironments, which may be linked to decreased connectivity and sociability. Furthermore, after a treatment with dietary curcumin, the abundance of certain bacterial taxa was greatly increasedand corresponded to increased myelination and white matter plasticity, contributing to improved sociability in Tsc2^+/− mice (Hsieh et al., 2024).

In this study, we characterized the gut microbiota of individuals with TSC and compared it to age- and sex-matched neurotypical controls and individuals with non-TSC epilepsy. A dietary survey was conducted to control for environmental influences, revealing no major differences among groups. Likewise, SCFA and BCFA levels did not differ significantly between groups.

Alpha-diversity metrics, which reflect the biodiversity within each sample, revealed no significant differences in species evenness or richness among the study groups. Regarding beta-diversity, although the differences did not reach statistical significance, we observed a trend toward clustering of TSC and EPI individuals, distinct from HC, consistent with existing literature suggesting an epilepsy-associated gut dysbiosis (Peng et al., 2018).

The taxonomic analysis highlighted a depletion of Firmicutes in both TSC and EPI, resulting in a decrease of F/B ratio, in agreement with literature studies (Zhu et al., 2024; Ceccarani et al., 2021). In our cohort, the observed decrease in Firmicutes appears to be primarily driven by a reduction in the Ruminococcaceae family, and at the genus level, by Gemmiger, and to a lesser extent, Faecalibacterium spp. Gemmiger has recently been identified as a biomarker of a healthy gut microbiota, noted for its anti-inflammatory properties. Together with Faecalibacterium and Roseburia, Gemmiger defines the three co-abundance groups (CAGs) identified in the healthy control group and considered beneficial due to their health-promoting activities (Forbes et al., 2018; Borghi et al., 2024), primarily through the production of SCFAs (Kircher et al., 2022). Notably, all three taxa are capable of producing butyrate - a metabolite known for its wide-ranging positive effects - including the ability to mitigate epileptogenic stimuli in rodent models by reducing oxidative stress and neuroinflammation (Adebayo et al., 2025; Li et al., 2021).

Recently published research demonstrated that active epilepsy in individuals with TSC is associated with elevated levels of GFAP compared to those with TSC but without epilepsy. This finding was confirmed in an external validation cohort and was also accompanied by increased levels of pro-inflammatory cytokines, including IL-17A, IL-17C, and TNF-α (Peng et al., 2018). Different microbial taxa in the gut exert either pro-inflammatory or anti-inflammatory effects and have been reported to modulate both local and systemic inflammation. Notably, Faecalibacterium and Roseburia are the most frequently reported for dampening the inflammation (Zhu et al., 2024). Their ability to modulate the Th17/Treg balance toward a more tolerogenic profile relies on their production of butyrate and its histone deacetylase (HDAC) inhibitory activity (Zhou et al., 2018).

Most of the alterations described in this exploratory study, including the above-mentioned depletion in Gemmiger and Faecalibacterium, were shared between TSC and EPI groups, suggesting that epilepsy may be the common underlying factor. However, some distinctions were noted: the TSC group exhibited an enrichment of Akkermansiaceae compared to both HC and EPI groups, while the EPI group showed a decreased relative abundance of Blautia_A and Faecousia. Increased abundance of Akkermansia, which to a lesser extent also involves the EPI group, has been observed in other neurological disorders, including epilepsy itself (Ceccarani et al., 2021), multiple sclerosis (Jangi et al., 2016), Alzheimer’s disease, and Parkinson’s disease (Fang et al., 2021). In the TSC group, Akkermansia was positively related to Bifidobacterium, one of the CAGs. In contrast, in the HC group, Akkermansia was associated with Faecalibacterium, suggesting potential alterations in the microbial network dynamics between taxa.

We observed a significant reduction of the genus Blautia in the EPI group compared with HC. Blautia includes species with diverse metabolic properties and, in turn, effects on human health (Liu et al., 2021), but the V3–V4 sequencing of the 16S rRNA gene does not allow for precise identification of the depleted species, underestimating the potential role in epilepsy. The EPI group was characterized by an enrichment of Faecousia, a recently described taxon belonging to the Oscillospiraceae family, with predicted capabilities for starch utilization and production of SCFAs (Hitch et al., 2025). Although Oscillospiraceae is generally considered a beneficial family, the significance of this finding remains difficult to interpret given the limited current knowledge (Yang et al., 2021).

Considering the whole spectrum of TANDs, many individuals with TSC in our cohort showed ID and/or neurodevelopmental disorder (ASD or ADHD). ASD is characterized by a distinct intestinal bacterial signature, and neuroinflammation has been proposed as an underlying mechanism. Indeed, increased intestinal permeability may pave the way to neuroinflammation via cytokines, leading to synaptic dysfunction and failure of microglia maturation (Hsiao et al., 2013). The bacterial phyla most frequently associated with higher inflammatory cytokine levels in ASD children are Prevotella, Bacteroidetes, and Bifidobacterium (Morton et al., 2023). Intriguingly, these genera were also enriched in our TSC group compared to HC and EPI, highlighting shared microbial signatures potentially associated with specific clinical features.

These preliminary findings, although derived from a small cohort, provide an important first step in clarifying the role of the microbiota–gut–brain axis (MGBA) in TSC. Larger, multi-center studies will be essential to confirm these results and to enable subgroup analyses based on epilepsy-related factors (e.g., duration, type and number of antiseizure medications) as well as neuropsychiatric profiles, which may yield more nuanced insights.

The sample size, while sufficient to identify broad trends, may limit the detection of more subtle associations. To ensure transparency, results close to conventional significance thresholds are reported with exact p-values and descriptive statistics.

Methodologically, the use of V3-V4 16S rRNA sequencing provides valuable taxonomic insight but restricts resolution at the genus level and does not capture microbial functional activity.

The gut microbiota plays a powerful role in shaping inflammation, which through the gut-brain axis may fuel epileptogenesis and worsen neurological symptoms in TSC. Given that the gut microbiota is both accessible and modifiable, investigating its potential role could offer promising avenues for the development of more personalized and effective treatments. Future studies with larger cohorts, longitudinal sampling, and multi-omics approaches will be necessary to confirm and expand upon these preliminary findings.