Detecting Alpha Synuclein Seeding Activity in Formaldehyde-Fixed MSA Patient Tissue by PMCA

Becker, Katelyn; Wang, Xinhe; Stel, Kayla Vander; Chu, Yaping; Kordower, Jeffrey H.; Ma, Jiyan

doi:10.1007/s12035-018-1007-y

Cited by 47 publications

(38 citation statements)

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“…The timeline of the experiment is represented in Figure 1a. The sizes of the PFFs were verified by transmission electron microscopy ( Figure 1b), which were consistent with the PFFs used in our previous studies [39].…”

Section: Increased Phosphorylated α-Syn Inclusions In Nsg Pffs Injectsupporting

confidence: 88%

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T cells limit accumulation of aggregate pathology following intrastriatal injection of α-synuclein fibrils

George¹,

Tyson²,

Rey

et al. 2020

Preprint

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α-Synuclein (α-syn) is the predominant protein in Lewy-body inclusions, which are pathological hallmarks of αsynucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA). Other hallmarks include activation of microglia, elevation of pro-inflammatory cytokines, as well as the activation of T and B cells.These immune changes point towards a dysregulation of both the innate and the adaptive immune system. T cells have been shown to recognize epitopes derived from α-syn and altered populations of T cells have been found in PD and MSA patients, providing evidence that these cells can be key to the pathogenesis of the disease.To study the role of the adaptive immune system with respect to α-syn pathology, we injected human α-syn preformed fibrils (PFFs) into the striatum of immunocompromised mice (NSG) and assessed accumulation of phosphorylated α-syn pathology as well as microgliosis. Compared to wildtype mice, NSG mice had an 8-fold increase in phosphorylated α-syn pathology in the substantia nigra. We also reconstituted T and B cell populations in the NSG mice with cells isolated from a wildtype mouse spleen. Interestingly, reconstituting the T cell population decreased the accumulation of α-syn pathology and resulted in persistent microgliosis when compared to non-transplanted mice; reconstitution of B cells did not alter the nigral neuropathology. Our work provides experimental evidence that T cells play a role in the pathogenesis of α-synucleinopathies.

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Section: Increased Phosphorylated α-Syn Inclusions In Nsg Pffs Injectsupporting

confidence: 88%

“…Recombinant human α-syn was purified similarly to this previously published protocol [38,39]. Briefly, the protein was expressed in BL21 E.coli transformed with a plasmid expressing human α-syn.…”

Section: Purification Of Recombinant α-Synuclein and Assembly Of Pre-mentioning

confidence: 99%

T cells limit accumulation of aggregate pathology following intrastriatal injection of α-synuclein fibrils

George¹,

Tyson²,

Rey

et al. 2020

Preprint

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“…We next tested whether 14-3-3 could regulate the seeding potential of ␣syn-enriched CM using the PMCA assay. PMCA is a technique initially developed to accelerate amplification of prion protein aggregates and has been adapted for measuring the growth of ␣syn aggregates (Herva et al, 2014;Shahnawaz et al, 2017;Becker et al, 2018). In this assay, recombinant ␣syn is incubated with minute amounts of potential misfolded ␣syn "seeds" in ␣syn-enriched CM to induce ␣syn polymerization; after a brief period of aggregation, the mixture is sonicated to break down the polymers into smaller sizes to generate more seeds for aggregation.…”

Section: -3-3 Reduces the Seeding Capability Of Released ␣Synmentioning

confidence: 99%

14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein

et al. 2018

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α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies. Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.

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“…However, since the lag-phase is a threshold value established by the investigator, the t50, named the time necessary to reach the 50% of the maximum fluorescence, is often used as a quantitative and objective measurement of the amplification process. The approximate linear relation between the t50 (or the lag-time) and the logarithm of the seed quantities has been shown for different pathogenic proteins like PrP sc ( 75 ) α-syn ( 4 , 7 , 76 ), Aβ1-40 ( 77 ), and Aβ1-42 ( 78 ). Sometimes, deviations from the ideal lineshape, like multiple inflection points or a decrease of the signal at the end of the reaction are present ( 6 , 79 ).…”

Section: Rt-quic and Pmca Assays: Basic Conceptsmentioning

confidence: 96%

Are We Ready for Detecting α-Synuclein Prone to Aggregation in Patients? The Case of “Protein-Misfolding Cyclic Amplification” and “Real-Time Quaking-Induced Conversion” as Diagnostic Tools

et al. 2018

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The accumulation and deposition of α-synuclein aggregates in brain tissue is the main event in the pathogenesis of different neurodegenerative disorders grouped under the term of synucleinopathies. They include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. To date, the diagnosis of any of these disorders mainly relies on the recognition of clinical symptoms, when the neurodegeneration is already in an advanced phase. In the last years, several efforts have been carried out to develop new diagnostic tools for early diagnosis of synucleinopathies, with special interest to Parkinson's disease. The Protein-Misfolding Cyclic Amplification (PMCA) and the Real-Time Quaking-Induced Conversion (RT-QuIC) are ultrasensitive protein amplification assays for the detection of misfolded protein aggregates. Starting from the successful application in the diagnosis of human prion diseases, these techniques were recently tested for the detection of misfolded α-synuclein in brain homogenates and cerebrospinal fluid samples of patients affected by synucleinopathies. So far, only a few studies on a limited number of samples have been performed to test PMCA and RT-QuIC diagnostic reliability. Neverthless, these assays have shown very high sensitivity and specificity in detecting synucleinopathies even at the pre-clinical stage. Despite the application of PMCA and RT-QuIC for α-synuclein detection in biological fluids is very recent, these techniques seem to have the potential for identifying subjects that will be likely to develop synucleinopathies.

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Detecting Alpha Synuclein Seeding Activity in Formaldehyde-Fixed MSA Patient Tissue by PMCA

Cited by 47 publications

References 50 publications

T cells limit accumulation of aggregate pathology following intrastriatal injection of α-synuclein fibrils

T cells limit accumulation of aggregate pathology following intrastriatal injection of α-synuclein fibrils

14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein

Are We Ready for Detecting α-Synuclein Prone to Aggregation in Patients? The Case of “Protein-Misfolding Cyclic Amplification” and “Real-Time Quaking-Induced Conversion” as Diagnostic Tools

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