Changes in the solubility and phosphorylation of α-synuclein over the course of Parkinson’s disease

Abstract

Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson's disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks. 500 mg of frozen putamen (affected preclinically) and frontal cortex (affected late) was homogenized, fractionated and α-synuclein levels evaluated using specific antibodies (syn-1, BD Transduction Laboratories; S129P phospho-α-synuclein, Elan Pharmaceuticals) and quantitative western blotting. Statistical analyses assessed the relationship between the different forms of α-synuclein, compared levels between groups, and determined any changes over the disease course. Soluble S129P was detected in controls with higher levels in putamen compared with frontal cortex. In contrast, insoluble α-synuclein occurred in Parkinson's disease with a significant increase in soluble and lipid-associated S129P, and a decrease in soluble frontal α-synuclein over the disease course. Increasing soluble S129P in the putamen correlated with increasing S129P in other fractions and regions. These data show that soluble non-phosphorylated α-synuclein decreases over the course of Parkinson's disease, becoming increasingly phosphorylated and insoluble. The finding that S129P α-synuclein normally occurs in vulnerable brain regions, and in Parkinson's disease has the strongest relationships to the pathogenic forms of α-synuclein in other brain regions, suggests a propagating role for putamenal phospho-α-synuclein in disease pathogenesis.

Modification of α-Synuclein by Phosphorylation: A Pivotal Event in the Cellular Pathogenesis of Parkinson’s Disease

“…The authors reported a progressive accumulation of pSyn-immunoreactive species in diseased brains compared to healthy controls, as well as a positive correlation between pSyn levels and the severity of disease symptoms. A similar study using brain samples from PD patients also reported a drastic accumulation of pSyn-positive inclusions in different brain regions at the late stages of the disease [128]. Together, these results suggest that the occurrence of pSyn is linked to the severity of disease progression.…”

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

“…This is a well-established technique to study aggregated α-syn 17, 18, 20, 21 a protein that is normally soluble in its native form but gain amyloidogenic or increased aggregation properties with disease progression or with genetic mutations. Nonetheless, some groups have used slight variations in SDS concentrations in their buffers (8% or 10% instead of 5%) 21,22,30 . The SDS is an anionic detergent and solubilises α-syn oligomers that can include membrane bound forms of α-syn, whereas urea, a chaotropic reagent denatures the insoluble aggregated and fibrillar or amyloidogenic forms of α-syn 20 .…”

“…The α-syn monomers were recognized to similar extents by all 4 antibodies although these can have variation depending on whether the α-syn antibody epitope is located either in the N-terminal or Cterminal 29 . Similarly, specific antibodies for phospho-alpha synuclein determine distinct high-molecular weight α-synuclein species 20,21 . In the protocol described here, the highly characterized antibody Syn-1 has been used [19][20][21] .…”

“…Similarly, specific antibodies for phospho-alpha synuclein determine distinct high-molecular weight α-synuclein species 20,21 . In the protocol described here, the highly characterized antibody Syn-1 has been used [19][20][21] .…”

“…Studies using post-mortem analysis of Parkinsonian brains have thus far provided important clues regarding normal and abnormal biology of α-syn both in sporadic PD and also PD associated with mutations in the LRRK2 gene [17][18][19][20][21][22] . Here, a biochemical extraction protocol (see scheme presented in Figure 1) for increasingly insoluble α-syn deposits from human post-mortem PD brain tissue that harbour α-syn aggregates to varying degrees has been described.…”