Omar M. A. El-Agnaf

Detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease

To date there is no accepted clinical diagnostic test for Parkinsons disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). α‐Synuclein (α‐syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding α‐syn in familial cases with early‐onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of α‐syn into insoluble aggregates. We recently reported the presence of α‐syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether α‐syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric “soluble aggregates” of α‐syn. Using this ELISA, we report the presence of significantly elevated (P=0.002) levels of oligomeric forms of α‐syn in plasma samples obtained from 34 PD patients compared with 27 controls; 52% (95% confidence intervals 0.353–0.687) of the PD patients displayed signals >0.5 OD with our ELISA assay in comparison to only 14.8% (95% confidence intervals 0.014 –0.281) for the control cases. An analysis of the tests diagnostic value revealed a specificity of 0.852 (95% confidence intervals 0.662– 0.958), sensitivity of 0.529 (95% confidence intervals 0.351–0.702) and a positive predictive value of 0.818 (95% confidence intervals 0.597–0.948). These observations offer new opportunities for developing diagnostic tests for PD and related diseases and for testing therapeutic agents aimed at preventing or reversing the aggregation of α‐syn.—El‐Agnaf, O. M. A., Salem, S. A., Paleologou, K. W., Curran, M. D., Gibson, M. J., Court, J. A., Schlossmacher, M. G., Allsop, D. Detection of oligomeric forms of α‐synuclein protein in human plasma as a potential biomarker for Parkinsons disease. FASEB J. 20, 419 –425 (2006)

α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma

Parkinsons disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of α‐synuclein protein (α‐syn) inside brain cells. It is likely that the formation of α‐syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding α‐syn have been found in inherited forms of PD. α‐Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of α‐syn in conditioned culture media from untransfected and α‐syn‐transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti‐α‐syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, α‐syn was identified as a single 15 kDa band, which co‐migrated with a recombinant form of the protein and reacted with five different antibodies to α‐syn. Our findings suggest that cells normally secrete α‐syn into their surrounding media, both in vitro and in vivo. The detection of extracellular α‐syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.

Aggregates from mutant and wild‐type α‐synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β‐sheet and amyloid‐like filaments

α‐Synuclein (α‐syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We have shown that human wild‐type α‐syn, mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. Here we report that aggregates of NAC and α‐syn proteins induced apoptotic cell death in human neuroblastoma SH‐SY5Y cells. These findings indicate that accumulation of α‐syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases.

Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century. Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past twenty years that the molecular events that precipitate disease have begun to be understood. Protein aggregation is a common feature of many neurodegenerative diseases, and it is assumed that the aggregation process plays a central role in pathogenesis. In this process, one molecule (monomer) of a soluble protein interacts with other monomers of the same protein to form dimers, oligomers, and polymers. Conformation changes in three-dimensional structure of the protein, especially the formation of β-strands, often accompany the process. Eventually, as the size of the aggregates increases, they may precipitate as insoluble amyloid fibrils, in which the structure is stabilized by the β-strands interacting within a β-sheet. In this review, we discuss this theme as it relates to the two most common neurodegenerative conditions—Alzheimer’s and Parkinson’s diseases.

Direct quantification of CSF α-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration

Because accumulation of alpha-synuclein (alphaS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several alphaS-derived peptides by mass spectrometry. The concentration of alphaS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total alphaS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF alphaS concentrations ranged from 0.8 to 16.2 pg/microl. Mean CSF alphaS values were lower in donors with a primary synucleinopathy (PD, DLB: n=57) than in the other two groups (AD, NCO: n=35; p=0.025). By contrast, living Creutzfeldt-Jakob disease patients showed markedly elevated CSF alphaS levels (n=8; mean, 300 pg/microl; p<0.001). Our results unequivocally confirm the presence of alphaS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF alphaS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF alphaS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies.

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Background: The oligomeric state of α-syn in vivo remains unknown. Results: α-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native α-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric α-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797–17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease

α‐Synuclein (α‐syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We report here that human wild‐type α‐syn, PD‐linked mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. The mutant α‐syn forms more β‐sheet and mature filaments than the wild‐type protein. These findings suggest that accumulation of α‐syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases.

A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders

Convergent biochemical and genetic evidence suggests that the formation of α‐synuclein (α‐syn) protein deposits is an important and, probably, seminal step in the development of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human α‐syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing α‐syn self‐aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7‐mer peptides spanning the entire α‐syn sequence, and identified amino acid residues 64‒100 of α‐syn as the binding region responsible for its self‐ association. Modified short peptides containing α‐syn amino acid sequences from part of this binding region (residues 69‒72), named α‐syn inhibitors (ASI), were found to interact with full‐ length α‐syn and block its assembly into both early oligomers and mature amyloid‐like fibrils. We also developed a cell‐permeable inhibitor of α‐syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α‐syn(A53T), a familial PD‐associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)‐induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl‐2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

Cerebrospinal fluid biomarkers in Parkinson disease

Clinical diagnosis of Parkinson disease (PD) is difficult in early stages of disease, with high risk of misdiagnosis. The long preclinical phase of PD provides the possibility for early therapeutic intervention once disease-modifying therapies have been developed, but lack of biomarkers for early diagnosis and monitoring of disease progression represents a major obstacle to achievement of this goal. Accordingly, research efforts aimed at identification of novel biomarkers have been increasing in the past 5 years. Cerebrospinal fluid (CSF) is an accessible source of brain-derived proteins, which mirror molecular changes that take place in the CNS. In this Review, we discuss evidence from numerous studies that have focused on identification of candidate CSF biomarkers for PD. Notably, molecular pathways related to α-synuclein, tau and β-amyloid peptides have received considerable attention. CSF levels of the protein DJ-1 are also of interest, although further investigation of this candidate marker is required. These studies support the usefulness of a combination of various CSF biomarkers of PD to increase diagnostic accuracy during early phases of the disease, and to differentiate PD from other neurodegenerative disorders.

Cerebrospinal fluid Tau/α-synuclein ratio in Parkinson's disease and degenerative dementias.

Although alpha‐synuclein is the main constituent of Lewy bodies, cerebrospinal fluid determination on its own does not seem fundamental for the diagnosis of synucleinopathies. We evaluated whether the combination of classical biomarkers, Aβ1–42, total tau, phosphorylated tau, and α‐synuclein can improve discrimination of Parkinsons disease, dementia with Lewy bodies, Alzheimers disease, and frontotemporal dementia. Aβ1–42, total tau, phosphorylated tau, and α‐synuclein were measured in a series of patients with Parkinsons disease (n = 38), dementia with Lewy bodies (n = 32), Alzheimers disease (n = 48), frontotemporal dementia (n = 31), and age‐matched control patients with other neurological diseases (n = 32). Mean α‐synuclein levels in cerebrospinal fluid were significantly lower in the pathological groups than in cognitively healthy subjects. An inverse correlation of α‐synuclein with total tau (r = −0.196, P < .01) was observed. In the group of patients with Parkinsons disease, Aβ1–42, total tau, and phosphorylated tau values were similar to controls, whereas total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios showed the lowest values. Cerebrospinal fluid α‐synuclein alone did not provide relevant information for Parkinsons disease diagnosis, showing low specificity (area under the curve, 0.662; sensitivity, 94%; specificity, 25%). Instead, a better performance was obtained with the total tau/α‐syn ratio (area under the curve, 0.765; sensitivity, 89%; specificity, 61%). Combined determination of α‐synuclein and classical biomarkers in cerebrospinal fluid shows differential patterns in neurodegenerative disorders. In particular, total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios can contribute to the discrimination of Parkinsons disease.