Currently uncurable, Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders. Characterised by progressive loss of dopaminergic neurons in the Substantia Nigra area in the brain, PD leads to the development of progressive motor and non-motor symptoms. Accumulation of α-synuclein (α-syn) aggregates is seen in the brain, which becomes neurotoxic when protein monomers combine to form insoluble amyloid fibrils. Any therapeutic strategies directed at inhibiting or reversing α-syn synthesis and its aggregation therefore present an opportunity for preventing PD disease from occurring or progressing.
Recent discoveries have implicated the gut microbiome in the progression and severity of PD. Gut bacteria have been proven to affect brain function by producing metabolites that enter the blood stream, influencing the immune response or modulating brain function. Microbiota from PD patients display notable differences compared to healthy controls, and fecal transplants from PD patients have been shown to exacerbate the symptoms of PD in mouse model, demonstrating that gut bacteria are not only a result of the disease but also have impact in its progression.
In this report authors have employed a bacterivore C.elegans model, including strains engineered to overexpressed human α-syn (strain NL5901), to study the effects of bacterium Bacillus subtilis PXN21 on α-syn aggregation.
Worms ectopically overexpressing, in muscle cells, human α-syn fused to fluorescence protein YFP, were fed with different bacterial diets made up of either a non-pathogenic strain of E.coli OP50 (control diet) or a stain of B.subtilis PXN21 (test diet), which was isolated from the commercially available probiotic product Bio-Kult (by ADM Protexin). Upon quantitative assessment of the number of microscopically visible aggregates of human α-syn-YFP protein, qPCR analysis of expression of unc-54 and α-syn transcripts and α-syn protein levels, together with assessment of monomeric/sub-monomeric forms of α-syn protein by non-denaturing gel electrophoresis, it has been shown that diet a containing B.subtilis led to inhibition of the aggregation of α-syn. Substantially lower numbers of aggregates were found in PXN21-fed worms compared to E.coli-fed controls. This effect was not caused by lowering the expression level of α-syn in PXN21-fed animals, as both higher levels of expression of α-syn gene and protein were observed in probiotic-fed C.elegans than control animals.
In addition, B.subtilis has shown an ability to inhibit α-syn aggregation throughout C.elegans' life span, despite consistently higher levels of α-syn transcript in the probiotic containing diet than in a E.coli diet. Interestingly the inhibitive effect of PXN21 bacterial stain on α-syn aggregation persisted for longer when worms were switched from a E.coli to PXN21 containing diet later in life (from L4 stage, the first larval stage) in comparison to worms fed continuously with PXN21 bacteria. This suggested that PXN21 probiotic changes the α-syn aggregated forms, possibly through cleavage or degradation. Larger quantities of monomeric/sub-monomeric form of α-syn were detected in protein extracts from PXN21-fed worms.
Furthermore, all B.subtilis stains tested extended the lifespan of α-syn-expressing transgenic animals. Although the mechanism of this effect is not fully understood, authors proposed that in older animals, it acted through formation of biofilms (a 3D bacterial community embedded in a self-produced extracellular matrix) and production of a nitric oxide (NO) metabolite. Both of those mechanisms were previously implicated in the increased the lifespan and stress tolerance of C.elegans.
All forms of bacterium including vegetative, metabolically active cells, dormant spores, and also dead B.subtilis (killed by combination of UV and antibiotics), were able to inhibit α-syn aggregation, even in young worms. This was despite the virtual inability of C.elegans to digest spores, and when no biofilm was present, and very limited levels of NO were available from digested bacteria in young worms. These observations suggested that novel, yet to be identified, active and stable metabolites must be associated with suppression of α-syn aggregation.
When authors tested a panel of laboratory B.subtilis strains, all strains showed similar effects on α-syn aggregation to the probiotic PXN21 stain following the continuous or food-switching diet regime. This suggested that the protective effect against α-syn aggregation is a general property of B.subtilis species.
To uncover the host response pathways that are modified by B.subtilis to include inhibitory effect on the aggregation of α-syn, global transcriptomics analysis by RNA sequencing was performed on sets of young adult animals fed on different diets: E.coli OP50, B.subtilis PXN21 and a mixed bacterial diet. Over 6,500 genes were differentially expressed in animals fed with B.subtilis compared to those fed on E.coli. The set contained a number of known suppressors of α-syn aggregation which were upregulated by the probiotic diet.
Among the top biological pathways that were upregulated by B.subtilis PXN21 were immune system processes, protein localisation and lipid metabolism, in particular genes involved in the sphingolipid metabolism pathway, known to modify α-syn pathology in PD. In particular, lagr-1 and asm-3 genes were upregulated while sptl-3 was downregulated. Analysis of α-syn aggregation in C.elegans loss-of-function mutants for the above genes upon B.subtilis diet, confirmed that lagr-1, asm-3 and sptl-3 genes had a direct effect on the aggregation of α-syn. All these C.elegans genes have corresponding orthologs in humans.
In summary, this study showed that alteration in sphingolipid metabolism triggered by a diet of B.subtilis PXN2, a probiotic strain that is available for human consumption, both inhibits aggregation and efficiently removes existing aggregates in C.elegans models with ectopic expression of human α-syn. Although in older worms, biofilm formation and NO production play a role in the reduction of α-syn aggregation, the protective effect seen earlier in life is independent of these mechanisms. Distinct metabolic states of the bacterium effect the α-syn aggregation through both dietary restriction (DR)-dependent and DR-independent mechanisms, which are still not fully understood, but worth investigating. The effect of B.subtilis on the nervous system, as well as its efficacy in mouse models of PD, presents promising avenues for further investigations.
The prospect of B.subtilis modifying α-syn aggregation in humans could open possibilities of diet-based, diseases-modifying interventions through the manipulation of microbiome composition in the gut or the development of drug therapies based on protective bacterial metabolites.
“Probiotic Bacillus subtilis Protects against α-Synuclein Aggregation in C.elegans”. Goya et al, 2020, Cell Reports 30, 367‑380