Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study

Yoshinaga, Saki; Yamanaka, Tatsuhiko; Miyazaki, Haruko; Okuzumi, Ayami; Hiyama, Akiko; Murayama, Shigeo; Nukina, Nobuyuki

doi:10.1016/j.bbrc.2019.11.142

Cited by 6 publications

(7 citation statements)

References 13 publications

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“…These authors emphasized that α-syn aggregates can only be detected following PK digestion. Therefore, the PK-resistant core of α-syn-containing structures was isolated to enable the analysis of the structure of α-syn aggregates using mass spectrometry [87].…”

Section: Discussionmentioning

confidence: 99%

Occurrence of Total and Proteinase K-Resistant Alpha-Synuclein in Glioblastoma Cells Depends on mTOR Activity

Ryskalin

Ferese

Morucci

et al. 2022

Cancers

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Alpha-synuclein (α-syn) is a protein considered to be detrimental in a number of degenerative disorders (synucleinopathies) of which α-syn aggregates are considered a pathological hallmark. The clearance of α-syn strongly depends on autophagy, which can be stimulated by inhibiting the mechanistic target of rapamycin (mTOR). Thus, the overexpression of mTOR and severe autophagy suppression may produce α-syn accumulation, including the proteinase K-resistant protein isoform. Glioblastoma multiforme (GBM) is a lethal brain tumor that features mTOR overexpression and severe autophagy inhibition. Cell pathology in GBM is reminiscent of a fast, progressive degenerative disorder. Therefore, the present work questions whether, as is analogous to neurons during degenerative disorders, an overexpression of α-syn occurs within GBM cells. A high amount of α-syn was documented in GBM cells via real-time PCR (RT-PCR), Western blotting, immunohistochemistry, immuno-fluorescence, and ultrastructural stoichiometry, compared with the amount of β- and γ-synucleins and compared with the amount of α-syn counted within astrocytes. The present study indicates that (i) α-syn is overexpressed in GBM cells, (ii) α-syn expression includes a proteinase-K resistant isoform, (iii) α-syn is dispersed from autophagy-like vacuoles to the cytosol, (iv) α-syn overexpression and cytosol dispersion are mitigated by rapamycin, and (v) the α-syn-related GBM-like phenotype is mitigated by silencing the SNCA gene.

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Section: Discussionmentioning

confidence: 99%

Occurrence of Total and Proteinase K-Resistant Alpha-Synuclein in Glioblastoma Cells Depends on mTOR Activity

Ryskalin

Ferese

Morucci

et al. 2022

Cancers

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“…Structural differences between brain-derived tau or α-syn aggregates and in vitro generated recombinant protein aggregates is an important issue to consider during interpretation of the data obtained with in vitro produced aggregates (Strohäker et al, 2019). We and others have recently demonstrated that protein aggregates generated by seeding of endogenous aggregates using the protein misfolding cyclic amplification (PMCA) assay maintains the conformational signatures of brain derived aggregates (Shahnawaz et al, 2020;Van der Perren et al, 2020;Yoshinaga et al, 2020). An array of fluorescent probes which effectively stain aggregates in brain sections, such as the ones developed in this study, can become a useful tool to screen for in vitro generated aggregates, which maintain the conformational fingerprint of their brain-derived counterparts.…”

Section: Discussionmentioning

confidence: 99%

Identification of Multicolor Fluorescent Probes for Heterogeneous Aβ Deposits in Alzheimer’s Disease

Mukherjee

Al-Lahham

Corkins

et al. 2022

Front. Aging Neurosci.

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Accumulation of amyloid-beta (Aβ) into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer’s disease (AD). There is a significant intra- and inter-individual variability in the morphology and conformation of Aβ aggregates, which may account in part for the extensive clinical and pathophysiological heterogeneity observed in AD. In this study, we sought to identify an array of fluorescent dyes to specifically probe Aβ aggregates, in an effort to address their diversity. We screened a small library of fluorescent probes and identified three benzothiazole-coumarin derivatives that stained both vascular and parenchymal Aβ deposits in AD brain sections. The set of these three dyes allowed the visualization of Aβ deposits in three different colors (blue, green and far-red). Importantly, two of these dyes specifically stained Aβ deposits with no apparent staining of hyperphosphorylated tau or α-synuclein deposits. Furthermore, this set of dyes demonstrated differential interactions with distinct types of Aβ deposits present in the same subject. Aβ aggregate-specific dyes identified in this study have the potential to be further developed into Aβ imaging probes for the diagnosis of AD. In addition, the far-red dye we identified in this study may serve as an imaging probe for small animal imaging of Aβ pathology. Finally, these dyes in combination may help us advance our understanding of the relation between the various Aβ deposits and the clinical diversity observed in AD.

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“…Slices were homogenized with disposable homogenizer SP and power masher II (Nippi, Inc., Tokyo, Japan). For sarkosyl soluble and insoluble fractions, the tissue homogenates were diluted in buffer (final; 10 mM Tris–HCl, pH 7.6, 0.8 M NaCl, 10% sucrose, 0.1% triton x-100, 50 mM NaF, 1 mM Na 3 VO 4 + cOmplete (Sigma-aldrich, 1 tablet/50 mL) as well as described previously 69 . 2% 2-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue were added to make final samples (protein concentration; 0.7 mg/mL).…”

Section: Methodsmentioning

confidence: 99%

“…Filter trap assay for detection the amount of insoluble high molecular weight α-syn aggregates were conducted using a HYBRI-DOT MANIFOLD (Cat: 1050MM, BRL Life Technologies Inc.) and cellulose acetate membrane filter with a pore size of 0.2μm (Cat: C020A142C, Advantec) as described previously 69 . In brief, the samples (5 μL) were mixed in 2% SDS and PBS and applied to apparatus.…”

Section: Methodsmentioning

confidence: 99%

Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission

et al. 2022

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Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.

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Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study

Cited by 6 publications

References 13 publications

Occurrence of Total and Proteinase K-Resistant Alpha-Synuclein in Glioblastoma Cells Depends on mTOR Activity

Occurrence of Total and Proteinase K-Resistant Alpha-Synuclein in Glioblastoma Cells Depends on mTOR Activity

Identification of Multicolor Fluorescent Probes for Heterogeneous Aβ Deposits in Alzheimer’s Disease

Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission

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