Studies On The Aggregation And Cytotoxicity Of Pathological C-terminal Truncated α-Synuclein

Parkinson’s disease(PD)is a kind of neurodegenerative disease that mainly affects the motor system,with a prevalence of 1% in people over 60 years of age.In 2015,there are 6.2 million patients suffering from PD worldwide.The main pathological features of PD are the death of dopaminergic neurons in the basal ganglia of the brain and the presence of eosinophilic Lewy bodies in neurons.Lewy bodies are brain amyloid deposits composed of fibrous aggregates formed by α-Synuclein(α-Syn).α-Syn is a 140 amino acid protein mainly found in the presynaptic terminals of the central nervous system and makes up as much as 1% of all proteins in the cytosol of brain cells.α-Syn is considered to be an intrinsically disordered protein in solution,which participates in the formation of SNARE complex and the functioning of the neuronal Golgi apparatus and vesicle trafficking.Under pathological conditions,α-Syn misfolds and aggregates into toxic intermidiates through intramolecular interaction,and finally forms fibrous deposits in the brain.Some single-nuclear acid mutants of α-Syn have been found in rare familial PD cases,such as A18 T,A29S,A30 P,E46K,H50 Q,G51D and A53 T.The aggregation and toxicity of these pathological mutants are not the same as those of wild-type α-Syn,and the relationships between these mutants and PD have not been fully illustrated.α-Syn undergoes a series of post-translational modifications(PTMs)in vivo,such as phosphorylation,nitration,ubiquitination,and proteolysis.Proteolysis generally occurs at the C-terminus of α-Syn,and about 10-30% of α-Syn in the Lewy bodies of PD patients is C-terminally truncated.Neurodegenerative diseases usually come up with chronic neuroinflammation.Caspase-1,which acts as an important regulator of neuroinflammation,has been reported to cleave α-Syn between Asp121 and Asn122 to generate a C-terminal truncated α-Syn 1-121(α-Syn121).Inhibition of Caspase-1 reduces inflammatory factor-induced cell death and alleviates α-Syn deposition and behavioral defects in mice with multiple system atrophy.Therefore,Caspase-1 and α-Syn121 may play important roles in the onset and/or pathogenisis of PD.What’s more,abnormal accumulation of metal ions(such as copper)has been reported in the Lewy bodies of PD patients,copper ions can generate reactive oxygen species through Fenton reaction,leading to cell death and tissue damage.It has been reported thatα-Syn can bind copper ions through N-terminal 1-9 amino acids and His50,thus inhibit the production of reactive oxygen species.Here we invesgated the aggregation,membrane disruption and cytotoxicity of full length α-Syn,the pathological mutants(A18T,A29 S,A30P,E46 K,H50Q,G51 D and A53T)and corresponding 1-121 truncated proteins.Moreover,we investigated the ability of α-Syn pathological mutants and the corresponding 1-121 truncated proteins in inhibiting copper-catalyzed production of reactive oxygen species.In section two,Th T fluorescence,cytotoxicity and dye leakage assays showed that α-Syn121 had accrelated aggregation kinetic and elevated cytotoxicity compared with full length α-Syn(α-Syn FL).However,the membrane disruption of α-Syn121 was weakened.TEM and FTIR assays demonstrated that α-Syn121 formed amorphous aggregates mainly composed of random coil structures,rather than linear fibers formed by α-Syn FL which is rich in b-sheet structures;and unlike low-toxic α-Syn FL aggregates,aggregates of α-Syn121 were still fatal to cells through inducing upregulated reactive oxygen species and apoptosis.In addition,through western blot and immunofluorescence assays,we also found that exogenous α-Syn121 aggregates could promote the activation of Caspase-1 to cleave endogenous α-Syn and generate α-Syn121,and induced α-Syn deposition in cells.In section three,Th T fluorescence,cytotoxicity and dye leakage assays showed that compared to full length α-Syn pathological mutants,the corresponding 1-121 truncated proteins had accrelated aggregation kinetic and elevated cytotoxicity,but weakened membrane disruption.Ascorbate consumption,3-CCA oxidation and Amplex red oxidation assays indicated that all of the 1-121 truncated proteins are weaker than the corresponding full-length proteins in inhibiting the production of reactive oxygen species.Taken together,our findings suggest a potential vicious circle in which aggregates of α-Syn121 activate Caspase-1 to cleave α-Syn to generate α-Syn121,while increased α-Syn121 forms amorphous aggregates rapidly,eventually lead to cell death and contribute to the onset and/or pathogenisis of PD.Meanwhile,the C-terminal 122-140 amino acids are also involved in the interaction of copper ions andα-Syn.C-terminal 19 amino acids truncation reduces the binding affinity betweenα-Syn and copper ions,which consequently improves the production of reactive oxygen species.Our results suggest that Caspase-1 andα-Syn121 may play important roles in the onset and/or pathogenisis of PD,and the clearance or inhibition of Caspase-1 andα-Syn121 may be beneficial to PD.