α-Synuclein aggregation is implicated in a variety of diseases including Parkinson's disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson’s disease. Both DJ-1 and α-synuclein, two proteins critically involved in Parkinson’s disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and α-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and α-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and α-synuclein from 117 patients with Parkinson’s disease, 132 healthy individuals and 50 patients with Alzheimer’s disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and α-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and α-synuclein levels were decreased in Parkinson’s patients versus controls or Alzheimer’s patients when blood contamination was controlled for. In the population aged ≥65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and α-synuclein, respectively, the sensitivity and specificity for patients with Parkinson’s disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for α-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or α-synuclein and the severity of Parkinson’s disease. Taken together, this represents the largest scale study for DJ-1 or α-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and α-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinson’s disease, if variables such as blood contamination and age are taken into consideration.

The presynaptic protein alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease. First, two missense mutations A30P and A53T cause inheritable early onset Parkinson's disease in some families. Secondly, alpha-synuclein is present in Lewy bodies of affected nerve cells in the predominant sporadic type of Parkinson's disease as well as in dementia with Lewy bodies. We demonstrate in the rat optic system that a portion of alpha-synuclein is carried by the vesicle-moving fast component of axonal transport and that it binds to rat brain vesicles through its amino-terminal repeat region. We find alpha-synuclein with the A30P mutation of familial Parkinson's disease devoid of vesicle-binding activity and propose that mutant alpha-synuclein may accumulate, leading to assembly into Lewy body filaments.

A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that α-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.

α-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in α-synuclein to autosomal dominant Parkinson’s disease and its presence in Lewy-like lesions. To gain insight into α-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by α-synuclein affinity chromatography of human brain cytosol. Direct binding assays using ¹²⁵I-labeled human tau40 demonstrated a reversible binding with a IC₅₀ about 50 pm. The interacting domains were localized to the C terminus of α-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and α-synuclein. Functionally, α-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that α-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed usingin vitrosystems are not generally seen to be associated with α-synucleinopathyin vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.