Tiago Fleming Outeiro

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.

Formation of toxic oligomeric α-synuclein species in living cells

Background Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinsons disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. Methodology/Principal Findings Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein. Conclusions/Significance This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.

CHIP Targets Toxic α-Synuclein Oligomers for Degradation

α-Synuclein (αSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric αSyn intermediates may represent the most toxic αSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on αSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced αSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic αSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of αSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric αSyn may contribute to the successful development of drug therapies that target oligomeric αSyn by mimicking or enhancing the powerful effects of CHIP.

Therapeutic role of sirtuins in neurodegenerative disease

The sirtuins are a family of enzymes which control diverse and vital cellular functions, including metabolism and aging. Manipulations of sirtuin activities cause activation of anti-apoptotic, anti-inflammatory, anti-stress responses, and the modulation of an aggregation of proteins involved in neurodegenerative disorders. Recently, sirtuins were found to be disease-modifiers in various models of neurodegeneration. However, almost in all instances, the exact mechanisms of neuroprotection remain elusive. Nevertheless, the manipulation of sirtuin activities is appealing as a novel therapeutic strategy for the treatment of currently fatal human disorders such as Alzheimers and Parkinsons diseases. Here, we review current data which support the putative therapeutic roles of sirtuin in aging and in neurodegenerative diseases and the feasibility of the development of sirtuin-based therapies.

Detection of novel intracellular α-synuclein oligomeric species by fluorescence lifetime imaging

Oligomerization and aggregation of α‐synuclein molecules are believed to play a major role in neuronal dysfunction and loss in Parkinsons disease (PD) and dementia with Lewy bodies. However, α‐synuclein oligomerization and aggregation have been detected only indirectly in cells using detergent extraction methods. Here, we show for the first time intracellular α‐synuclein oligomerization using fluorescence lifetime imaging (FLIM). Two forms of α‐synuclein homomeric interactions were detected: an antiparallel amino terminus‐carboxyl terminus interaction between α‐synuclein molecules, and a close amino terminuscarboxy terminus interaction within single α‐synuclein molecules. Coexpression of the chaperone protein Hsp70, which can block α‐synuclein toxicity in several systems, causes α‐synuclein to adopt a different, open conformation, but Hsp70 does not alter α‐synuclein–synuclein interactions. Thus, the neuroprotective effect of Hsp70 can be explained by its chaperone activity on α‐synuclein molecules, rather than alteration of α‐synuclein–α‐synuclein interactions.—Klucken, J., Outeiro, T. F., Nyugen, P., McLean, P. J., and Hyman, B. T. Detection of novel intracellular α‐synuclein oligomeric species by fluorescence lifetime imaging. FASEB J. 20, 2050–2057 (2006)

Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.

Dopamine-Induced Conformational Changes in Alpha-Synuclein

Background Oligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinsons disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7]. Methodology/Principal Findings Here, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species. Conclusion/Significance Our results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinsons disease.

10 – Protein Aggregation Disorders

Protein aggregation disorders are a group of diseases that arise because of or are associated with the misfolding and aggregation of one or more proteins. They are believed to result from the failure of proteins to reach their active state or from the accumulation of abnormally folded proteins. Despite the strong connection between protein misfolding and aggregation and disease, the manner by which it results in disease is still unknown. The “amyloid hypothesis” states that the aggregation of proteins into an ordered fibrillar structure is causally related to aberrant protein interactions that culminate in neuronal dysfunction and ultimately neurodegeneration. Evidence from a variety of diseases suggests that specific alterations in the primary sequence of cellular proteins, posttranslational modifications, or defective interactions with other proteins can impose conformational constraints that alter the normal function/biology of these proteins and facilitate aggregation. Protein aggregation disorders include Alzheimers disease (AD), synucleinopathies [Parkinsons disease (PD)], and prion diseases. Accumulation of Aβ in the brain is the primary culprit of AD-related pathogenesis, including neurofibrillary tangles (NFT) formation, synapse loss, and neuronal cell death. In PD, the disconnection between pathogenesis and α-synuclein inclusion formation adds support for roles of α-synuclein oligomers in toxicity and pathogenesis. The accumulation of PrP Sc (prion protein) in the brain is the hallmark of prion diseases, but, as in other neurodegenerative diseases, whether it is directly responsible for the devastating pathology is still unclear.