Disease-associated Alpha-synuclein Research Articles

Discrimination of MSA-P and MSA-C by RT-QuIC analysis of olfactory mucosa: the first assessment of assay reproducibility between two specialized laboratories

BackgroundDetection of the pathological and disease-associated alpha-synuclein (αSynD) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson’s disease (PD). We recently showed that αSynD can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes.MethodsOM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data.ResultsThe αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83–1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability.ConclusionsOur study provides evidence that OM-αSynD may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.

Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson\u2019s disease and dementia with Lewy bodies

Definitive diagnosis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

Deposits of disease-associated alpha-synuclein may be present in the dura mater in Lewy body disorders: implications for potential inadvertent transmission by surgery.

Deposition of alpha-synuclein in the brain is a hallmark of Lewy body disorders. Alpha-synuclein has been considered to show prion-like properties. Prion diseases can be transmitted by the transplantation of cadaveric dura mater causing iatrogenic Creutzfeldt-Jakob disease. Recent observations of amyloid-β deposition in dural grafts support the seeding properties of amyloid-β. Here we assessed the presence of alpha-synuclein in dura mater samples as a potential transmissible seed source. We immunostained 32 postmortem dura mater samples; 16 cases with Lewy-body disorder (LBD) showing different pathology stages and 16 non-LBD cases for phosphorylated (Ser129) and disease-associated (5G4) alpha-synuclein. Disease-associated alpha-synuclein aggregates were identified in intradural nerve fibres and associated with a vessel in a single LBD-Braak stage 4 case. We conclude that alpha-synuclein is detectable, although rarely, in dura mater samples in patients with LBD. The risk of potential transmissibility of dural alpha-synuclein deserves assessment by complementary experimental studies.

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.

Tissue transglutaminase modulates α‐synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.