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Cell-Based Screen Using Amyloid Mimic ?23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of ?-Synuclein and Huntingtin Aggregates.

ABSTRACT: Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (?23) expression model to screen potential lead compounds inhibiting ?23-induced toxicity. Highthroughput screening identified several natural compounds as nuclear ?23 inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic ?23 aggregates and protects SH-SY5Y cells from toxicity induced by ?23 expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and ?-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and ?-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited ?-synuclein aggregation but also disaggregated preformed ?-synuclein fibrils in vitro . Taken together, our results suggest that a Tet-Off ?23 cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

SUBMITTER: Ham S

PROVIDER: S-EPMC6602149 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Cell-Based Screen Using Amyloid Mimic β23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of α-Synuclein and Huntingtin Aggregates.

Ham Sangwoo S Kim Hyojung H Hwang Seojin S Kang Hyunook H Yun Seung Pil SP Kim Sangjune S Kim Donghoon D Kwon Hyun Sook HS Lee Yun-Song YS Cho MyoungLae M Shin Heung-Mook HM Choi Heejung H Chung Ka Young KY Ko Han Seok HS Lee Gum Hwa GH Lee Yunjong Y

Molecules and cells 20190601 6

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (β23) expression model to screen po ...[more]

PMID: 31250621

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Project description:BackgroundAlzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear.MethodsThe aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space.ResultsOur data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte.ConclusionsTaken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Project description:Parkinson's disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPP+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+. This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.

Dataset Information

Cell-Based Screen Using Amyloid Mimic ?23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of ?-Synuclein and Huntingtin Aggregates.

Publications

Cell-Based Screen Using Amyloid Mimic β23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of α-Synuclein and Huntingtin Aggregates.

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