Mechanistic Insight Into the Role of Gut Bacterial Functional Amyloids in Modulating -synuclein Aggregation

Parkinson’s disease (PD) is a neurodegenerative disease affecting nearly 1 million people in the United States leading to a total estimated financial burden of $52 billion. The hallmarks of PD include the amyloidogenic aggregation of α-synuclein, loss of motor function and loss of dopaminergic neurons in the substantia nigra. Symptoms occur late in the course of disease and the current treatment is focused mainly on alleviating symptoms rather that preventing the disease progression. Interestingly, nearly 80% of PD patients experience gastrointestinal dysfunction like constipation, which may precede the onset of motor symptoms by years. Moreover, many studies have shown that there is a significant difference in the gut microbiome between PD patients and healthy individuals. Coupled with the early gastrointestinal dysfunction, these studies have led to the speculations that the gut microbiome may be implicated in PD. The direct evidence of gut microbiome’s role in PD was provided by a recent study in which colonization α-synuclein over-expressing (AOE) mice gut with E. coli expressing a functional amyloid, CsgA lead to enhanced PD associated pathophysiology. Moreover, the in vitro amyloidogenic aggregation of α-synuclein was accelerated by E. coli CsgA. In addition to E. coli, the human gut is home to diverse species of microorganism with potential CsgA producing microorganism. The impact of gut bacterial CsgA on α-synuclein aggregation and the mechanism behind the accelerated α-synuclein aggregation is still poorly understood. My work has identified CsgA homologs from diverse human gut bacterial species and characterized their aggregation kinetics. Gut bacterial CsgA homologs display diverse aggregation kinetics and differentially modulate α-synuclein aggregation. Interestingly, slow aggregating CsgA homologs have enhanced ability to accelerate α-synuclein compared to fast aggregating CsgA homologs. In addition to this, slow aggregating CsgA homologs form stable dimeric species compared to fast aggregating CsgA homologs and the stability of these dimeric species is correlated their ability to accelerate α-synuclein aggregation. I also report a complex between CsgA and α-synuclein that serves as a “seed” to facilitate acceleration of α-synuclein aggregation. Since the differential aggregation kinetics of gut bacterial CsgA homologs governs their ability to accelerate α-synuclein aggregation, I also address the sequence determinants of amyloidosis of gut bacterial CsgA homologs. There are certain “gatekeeper” amino acid residues in CsgA which inhibit the intrinsic amyloid formation propensity of CsgA. In addition to the earlier described negatively charged aspartic acid (D) residues, I identified new positively charged arginine (R) and lysine (K) gatekeeper residues in CsgA and demonstrate the molecular mechanism governing CsgA amyloid formation. The electrostatic interactions between the positively (R and K) and negatively charged (D) gatekeeper residues determine the nucleation rate of CsgA amyloid formation. Salt mediated charge masking or pH induced charge neutralization/reversal severely affected the inhibition of the intrinsic amyloid formation propensity of CsgA. Taken together, my work reveals the mechanism behind the role of gut bacterial functional amyloid in PD causation. Additionally, my work also provides molecular insights into gut bacterial functional amyloidogenesis paving the way to developing future therapeutics to prevent and/or treat PD.PhDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174365/1/sbhoite_1.pd