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Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression.

ABSTRACT: Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (?Syn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study ?Syn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different ?Syn constructs: ?Syn, ?Syn coupled to the fluorescence protein Venus (?Syn-Venus), and ?Syn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of ?Syn upon incubation with DMSO and FeCl₃ along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly ?Syn-overexpressing cell lines holding high potential for the screening for modulators of ?Syn aggregation and ?Syn-mediated toxicity.

SUBMITTER: Bartels M

PROVIDER: S-EPMC6591385 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression.

Bartels Martin M Weckbecker Daniel D Kuhn Peer-Hendrik PH Ryazanov Sergey S Leonov Andrei A Griesinger Christian C Lichtenthaler Stefan F SF Bötzel Kai K Giese Armin A

Scientific reports 20190624 1

Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (αSyn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study αSyn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different αSyn constructs: αSyn, αSyn coupled to the fluorescence protein Venus (αSyn-Venus), and αSyn coupled to ...[more]

PMID: 31235814

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Project description:Alpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies. αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal counts in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.

Dataset Information

Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression.

Publications

Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression.

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