Antibody-based methods for the measurement of α-synuclein concentration in human cerebrospinal fluid - method comparison and round robin study.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF ('total' α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83-0.99), and good correlation with each other (R2 0.64-0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays.

Switch-peptides: design and characterization of controllable super-amyloid-forming host-guest peptides as tools for identifying anti-amyloid agents.

Several amyloid-forming proteins are characterized by the presence of hydrophobic and highly amyloidogenic core sequences that play critical roles in the initiation and progression of amyloid fibril formation. Therefore targeting these sequences represents a viable strategy for identifying candidate molecules that could interfere with amyloid formation and toxicity of the parent proteins. However, the highly amyloidogenic and insoluble nature of these sequences has hampered efforts to develop high-throughput fibrillization assays. Here we describe the design and characterization of host-guest switch peptides that can be used for in vitro mechanistic and screening studies that are aimed at discovering aggregation inhibitors that target highly amyloidogenic sequences. These model systems are based on a host-guest system where the amyloidogenic sequence (guest peptide) is flanked by two beta-sheet-promoting (Leu-Ser)(n) oligomers as host sequences. Two host-guest peptides were prepared by using the hydrophobic core of Abeta comprising residues 14-24 (HQKLVFFAEDV) as the guest peptide with switch elements inserted within (peptide 1) or at the N and C termini of the guest peptide (peptide 2). Both model peptides can be triggered to undergo rapid self-assembly and amyloid formation in a highly controllable manner and their fibrillization kinetics is tuneable by manipulating solution conditions (for example, peptide concentration and pH). The fibrillization of both peptides reproduces many features of the full-length Abeta peptides and can be inhibited by known inhibitors of Abeta fibril formation. Our results suggest that this approach can be extended to other amyloid proteins and should facilitate the discovery of small-molecule aggregation inhibitors and the development of more efficacious anti-amyloid agents to treat and/or reverse the pathogenesis of neurodegenerative and systemic amyloid diseases.

E46K Parkinson's-linked mutation enhances C-terminal-to-N-terminal contacts in alpha-synuclein.

Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein alpha-synuclein (alphaS) in neurons. Intramolecular contacts between the acidic C-terminal tail of alphaS and its N-terminal region have been proposed to regulate alphaS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked alphaS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that C-terminal-to-N-terminal contacts in alphaS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate alphaS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).

Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein.

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?

Amyloid-beta (Abeta) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Abeta. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Abeta on cultured cells; in particular, the evidence suggests that Abeta-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Abeta(1-40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that Abeta(1-40) induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADP-ribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Abeta(1-40) inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Abeta-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1beta plays a significant role in mediating the effects of Abeta(1-40) because Abeta(1-40) increased hippocampal IL-1beta and because several effects of Abeta(1-40) were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Abeta-induced changes in hippocampal plasticity are likely to be dependent upon IL-1beta-triggered activation of JNK.