The Coordinated Action of Calcineurin and Cathepsin D Protects Against α-Synuclein Toxicity

Aufschnaiter, Andreas; Habernig, Lukas; Kohler, Verena; Diessl, Jutta; Carmona-Gutiérrez, Didac; Eisenberg, Tobias; Keller, Walter; Büttner, Sabrina

doi:10.3389/fnmol.2017.00207

Cited by 23 publications

(30 citation statements)

References 78 publications

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“…Yeast strains and culture conditions S. cerevisiae strains used in this study were all derived from BY4741 (MATa; his3∆1; leu2∆0, met15∆0; ura3∆0, Euroscarf). CRZ1 (BY4741 crz1::hphNT1) and CNB1 (BY4741 cnb1::hphNT1) deletion mutants have been described previously [19]. The PMR1 deletion mutant was generated via homologous recombination following established protocols [20].…”

Section: Methodsmentioning

confidence: 99%

“…Cell death analysis, epifluorescence microscopy and flow cytometry PI staining as an indicator of loss of membrane integrity was used to assess cell death and was essentially performed as previously described [19]. In brief, approximately 1x10 6 cells per sample were harvested in 96-well plates and incubated for 5 min with PI (81845, Sigma), final concentration 500 ng/ml in phosphate buffered saline (25 mM potassium phosphate, 0.9% NaCl; adjusted to pH 7.2).…”

Section: Immunoblot Analysismentioning

confidence: 99%

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Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

Diessl¹,

Nandy²,

Schug³

et al. 2020

Microb Cell

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The protein phosphatase calcineurin is activated in response to rising intracellular Ca 2+ levels and impacts fundamental cellular processes in organisms ranging from yeast to humans. In fungi, calcineurin orchestrates cellular adaptation to diverse environmental challenges and is essential for virulence of pathogenic species. To enable rapid and large-scale assessment of calcineurin activity in living, unperturbed yeast cells, we have generated stable and destabilized GFP transcriptional reporters under the control of a calcineurin-dependent response element (CDRE). Using the reporters, we show that the rapid dynamics of calcineurin activation and deactivation can be followed by flow cytometry and fluorescence microscopy. This system is compatible with live/dead staining that excludes confounding dead cells from the analysis. The reporters provide technology to monitor calcineurin dynamics during stress and ageing and may serve as a drug-screening platform to identify novel antifungal compounds that selectively target calcineurin.-catalytic subunit of CN, CnB -regulatory subunit of CN, GFP -green fluorescent protein, PI -propidium iodide. J. Diessl et al. (2020) Calcineurin activity in living yeast OPEN ACCESS | www.microbialcell.com 107 Microbial Cell | APRIL 2020 | Vol. 7 No. 4 FIGURE 1: GFP and GFP PEST function as reporters of calcineurin activity. (A) Schematics of pAMS366-4XCDRE-lacZ, pAMS366-4xCDRE-GFP and pAMS366-4xCDRE-GFP PEST plasmids encoding reporters for CN activity. (B) CN activity was determined via β-gal activity or via flow cytometric quantification of GFP fluorescence intensities in exponentially growing wild type and ∆crz1 cells equipped with either pAMS366-4xCDRE-lacZ, pAMS366-4xCDRE-GFP or pAMS366-4xCDRE-GFP PEST . Values are shown as fold of ∆crz1 cells. Means ± SEM, n = 4. (C) CN activity was measured as in (B) in exponentially growing wild type, ∆pmr1, ∆cnb1 and ∆crz1 cells equipped with the indicated reporter plasmid.For stimulation of CN activity, cells were treated with 50 mM Ca 2+ 1 h prior to measurement. Fold of untreated wild type cells is shown. Dead cells were excluded from the analysis via propidium iodide (PI) staining. Means ± SEM, n = 4. (D) Representative immunoblots of protein extracts from cells described in (C). Immunoblots were decorated with antibodies against β-gal or GFP, respectively, and Pgk1 as loading control. (E-G) Histograms of cells quantified in (C) indicating the shift in green fluorescence intensity of wild type cells with and without 50 mM Ca 2+ treatment and ∆pmr1 cells equipped with the GFP (E) or the GFP PEST reporter (F, G).

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Section: Methodsmentioning

confidence: 99%

Section: Immunoblot Analysismentioning

confidence: 99%

Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

Diessl¹,

Nandy²,

Schug³

et al. 2020

Microb Cell

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“…Loss of membrane integrity was determined with propidium iodide (PI) staining as previously described (Aufschnaiter et al, 2017 ). In brief, 2*10 6 cells were harvested in 96-well plates at indicated time points and resuspended in 250 μl of phosphate buffered saline (PBS, 25 mM potassium phosphate, 0.9% NaCl; adjusted to pH 7.2) containing 100 μg/l PI.…”

Section: Methodsmentioning

confidence: 99%

The Enzymatic Core of the Parkinson’s Disease-Associated Protein LRRK2 Impairs Mitochondrial Biogenesis in Aging Yeast

Aufschnaiter

Kohler

Walter

et al. 2018

Front. Mol. Neurosci.

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Mitochondrial dysfunction is a prominent trait of cellular decline during aging and intimately linked to neuronal degeneration during Parkinson’s disease (PD). Various proteins associated with PD have been shown to differentially impact mitochondrial dynamics, quality control and function, including the leucine-rich repeat kinase 2 (LRRK2). Here, we demonstrate that high levels of the enzymatic core of human LRRK2, harboring GTPase as well as kinase activity, decreases mitochondrial mass via an impairment of mitochondrial biogenesis in aging yeast. We link mitochondrial depletion to a global downregulation of mitochondria-related gene transcripts and show that this catalytic core of LRRK2 localizes to mitochondria and selectively compromises respiratory chain complex IV formation. With progressing cellular age, this culminates in dissipation of mitochondrial transmembrane potential, decreased respiratory capacity, ATP depletion and generation of reactive oxygen species. Ultimately, the collapse of the mitochondrial network results in cell death. A point mutation in LRRK2 that increases the intrinsic GTPase activity diminishes mitochondrial impairment and consequently provides cytoprotection. In sum, we report that a downregulation of mitochondrial biogenesis rather than excessive degradation of mitochondria underlies the reduction of mitochondrial abundance induced by the enzymatic core of LRRK2 in aging yeast cells. Thus, our data provide a novel perspective for deciphering the causative mechanisms of LRRK2-associated PD pathology.

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“… 2006 ), as well as reduced vacuolar proteolytic function (Aufschnaiter et al. 2017 ) and results in cytotoxicity, which involves the Golgi-localized Ca 2+ transporter, Pmr1p, and the mitochondrial nuclease, Nuc1p (yeast homolog of EndoG). Importantly, these mechanisms are conserved in multicellular organisms and neuroblastoma cell lines, respectively, consolidating the use of yeast in elucidating disease mechanisms (Büttner et al .…”

Section: Yeast Chemogenomic Screens Against Age-related Diseasesmentioning

confidence: 99%

Yeast as a tool to identify anti-aging compounds

Zimmermann

Hofer

Pendl

et al. 2018

FEMS Yeast Research

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In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae, has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification.

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The Coordinated Action of Calcineurin and Cathepsin D Protects Against α-Synuclein Toxicity

Cited by 23 publications

References 78 publications

Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

The Enzymatic Core of the Parkinson’s Disease-Associated Protein LRRK2 Impairs Mitochondrial Biogenesis in Aging Yeast

Yeast as a tool to identify anti-aging compounds

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