Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates.

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

Heterogeneity in α-synuclein subtypes and their expression in cortical brain tissue lysates from Lewy body diseases and Alzheimer's disease.

AIMS: Lewy body diseases are neuropathologically characterized by the abnormal accumulation of α-synuclein (α-syn) protein within vulnerable neurons. Although studies have evaluated α-syn in post mortem brain tissue, previous findings have been limited by typically employing pan-α-syn antibodies that may not recognize disease-relevant forms of protein. We investigated the presence of α-syn species present in post mortem brain tissues from Lewy body disease and Alzheimer's disease. METHODS: Soluble and insoluble/aggregated α-syn from frontal cortex of post mortem brain tissues form Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD) and aged control cases were sequentially extracted using buffers with increasing detergent concentrations. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and pS129-α-syn). ELISA data were validated by western blot and compared to histological data from the same region of the contralateral hemisphere. RESULTS: There was no difference in t-α-syn levels between groups in the aqueous-soluble, detergent-soluble or urea-soluble tissue fractions. However, aqueous-soluble non-phosphorylated o-α-syn was increased not only in PD and DLB but also in AD without neocortical Lewy bodies. In PD and AD, pS129-α-syn was increased in the detergent-soluble tissue fragment and, in AD, this was positively correlated with the burden of tau pathology. Increased levels of urea-soluble pS129-α-syn were demonstrated only in DLB tissue lysates but this did not correlate with Lewy body pathological burden. CONCLUSIONS: Taken together, these findings suggest that DLB have elevated levels of insoluble pS129-α-syn, but that increased levels of aqueous-soluble o-α-syn and detergent-soluble pS129-α-syn are also observed in PD and AD, suggesting different changes to α-syn across the spectrum of neurodegenerative proteopathies.

Dynamic modeling of α-synuclein aggregation in dopaminergic neuronal system indicates points of neuroprotective intervention: experimental validation with implications for Parkinson's therapy.

Protein aggregation is the major pathological hallmark seen in neurodegenerative disorders such as Parkinson's disease (PD). Alpha-synuclein (αS) is the main component of protein aggregates that form Lewy bodies (LBs) in PD and dementia with LBs. There have been several attempts to intervene in the process of expression, modification, clearance, and aggregation of αS as a therapeutic strategy toward neuroprotection. In this study, we have employed a novel, predictive, system level approach in silico to study four different strategies of anti-aggregation therapies: (a) reduction in αS modifications such as phosphorylation, nitration, or truncation in an approach called "seed clearance;" (b) "anti-oligomerization" approach through blocking the early oligomers formation; (c) "oligomers clearance" process by increasing its lysosomal degradation; and (d) "anti-aggregation" that involves prevention of aggregate formation at a later stage. These strategies were tested in a virtual dopaminergic neuronal system triggered by overexpression (OE) of mutant αS-A53T with or without rotenone (Rot)-induced oxidative stress. The results were compared by analyzing markers related to various end points such as oxidative stress, dopamine (DA) metabolism, proteasome function, survival and apoptosis. The experimental system and anti-oligomerization strategies were recapitulated in vitro in M17 dopaminergic cells overexpressing mutant αS-A53T triggered with Cu(II)-mediated oxidative stress, and the experimental data prospectively corroborated with the predictive results. Through this analysis, we found that intervention in the early part of the aggregation pathway by prevention of oligomer formation and increased clearance is indeed a good neuroprotective strategy, whereas anti-aggregation efforts to break up the aggregate at later stages has negative effects on the system.

Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease.

BACKGROUND: To date, there is no accepted clinical diagnostic test for Parkinson disease (PD) that is based on biochemical analysis of blood or CSF. The discovery of mutations in the SNCA gene encoding α-synuclein in familial parkinsonism and the accumulation of α-synuclein in the PD brain suggested a critical role for this protein in PD etiology. METHODS: We investigated total and α-synuclein oligomers levels in CSF from patients clinically diagnosed with PD, progressive supranuclear palsy (PSP), or Alzheimer disease (AD), and age-matched controls, using ELISA developed in our laboratory. RESULTS: The levels of α-synuclein oligomers and oligomers/total-α-synuclein ratio in CSF were higher in the PD group (n = 32; p < 0.0001, Mann-Whitney U test) compared to the control group (n = 28). The area under the receiver operating characteristic curve (AUC) indicated a sensitivity of 75.0% and a specificity of 87.5%, with an AUC of 0.859 for increased CSF α-synuclein oligomers in clinically diagnosed PD cases. However, when the CSF oligomers/total-α-synuclein ratio was analyzed, it provided an even greater sensitivity of 89.3% and specificity of 90.6%, with an AUC of 0.948. In another cross-sectional pilot study, we confirmed that the levels of CSF α-synuclein oligomers were higher in patients with PD (n = 25) compared to patients with PSP (n = 18; p < 0.05) or AD (n = 35; p < 0.001) or control subjects (n = 43; p < 0.05). CONCLUSION: Our results demonstrate that levels of α-synuclein oligomers in CSF and the oligomers/total-α-synuclein ratio can be useful biomarkers for diagnosis and early detection of PD.

Protein aggregation, metals and oxidative stress in neurodegenerative diseases.

There is clear evidence implicating oxidative stress in the pathology of many different neurodegenerative diseases. ROS (reactive oxygen species) are the primary mediators of oxidative stress and many of the aggregating proteins and peptides associated with neurodegenerative disease can generate hydrogen peroxide, a key ROS, apparently through interactions with redox-active metal ions. Our recent results suggest that ROS are generated during the very early stages of protein aggregation, when protofibrils or soluble oligomers are present, but in the absence of mature amyloid fibrils. The generation of ROS during early-stage protein aggregation may be a common, fundamental molecular mechanism underlying the pathogenesis of oxidative damage, neurodegeneration and cell death in several different neurodegenerative diseases. Drugs that specifically target this process could be useful in the future therapy of these diseases.

Alpha-synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy.

There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which alpha-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of alpha-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce alpha-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-synuclein (A53T), a familial PD-associated mutation.

Structure and neurotoxicity of novel amyloids derived from the BRI gene.

A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.g. Alzheimer's disease (beta-amyloid) and Parkinson's disease (alpha-synuclein). Other examples are rare familial dementias which involve the BRI gene. In a British family, mutation of the termination codon extends the reading frame of BRI to yield a furin-processed 34-residue peptide (Abri; British dementia peptide), 11 residues longer than the wild-type (WT). In a Danish family, a ten-base insertion also yields a 34-residue peptide (Adan; Danish dementia peptide). To explore the roles of Abri and Adan in neurodegeneration, we synthesized Abri and Adan in oxidized and reduced forms and generated transgenic mice colonies expressing the WT and mutated forms of BRI. We have generated transgenic mice colonies bearing the genes coding for WT-BRI, Adan and Abri under the control of the Thy1 promoter. Whereas WT-BRI transgenic mice express full-length WT-BRI protein in their brains, Adan protein is fully processed to small peptides.

Aggregation and neurotoxicity of alpha-synuclein and related peptides.

Fibrillar deposits of alpha-synuclein occur in several neurodegenerative diseases. Two mutant forms of alpha-synuclein have been associated with early-onset Parkinson's disease, and a fragment has been identified as the non-amyloid-beta peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of alpha-synuclein and NAC change conformation to beta-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8-18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Hence residues 8-16 of NAC comprise the region crucial for toxicity. Toxicity induced by alpha-synuclein, NAC and NAC(1-18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.