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Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Mitochondrial dysfunction and Neurotoxicity||KeyEvent||Cataia Ives (send email)||Open for citation & comment||TFHA/WNT Endorsed|
|Mitochondrial complex inhibition leading to liver injury||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
Key Event Description
The concept of proteostasis refers to the homeostasis of proteins in space and time, i.e. the correct balance between protein synthesis, modification, transport and degradation. Disturbance of proteostasis results in pathological changes either by loss of function events (lack of a pivotal protein/protein function) or by a gain of undesired functions (aggregation of a protein leading to the formation of inclusions and new structures in cells and disturbing turnover of many unrelated proteins).
Proteostasis regulation is the main defence mechanism against toxic proteins, whose accumulation could greatly compromise normal cellular function and viability. Therefore, the chaperone and degradation systems assuring the removal of misfolded and aggregated proteins, as well as damaged, dysfunctional cellular organelles (e.g. defective mitochondria) play a key role in cellular homeostasis (Lee et al., 2012). The two major degradation systems are the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP) (Korolchuk et al., 2010; Kroemer et al., 2010; Ravikumar et al., 2010). The UPS works through the attachment of multiple ubiquitin molecules to a protein substrate, followed by the subsequent degradation of the tagged polyubiquitinated protein by the proteasome (Ciechanover, 1998; Ciechanover and Brundin, 2003). A compromised function of the UPS leads to the accumulation of ubiquitylated proteins, such as α-synuclein (Ii et al. 1997; Spillantini et al. 1997; Sulzer and Zecca 2000). The accumulation of polyubiquitinated proteins, as a consequence of a dysfunctional proteasome activity, is observed in some pathologies, and experimental inhibition of the proteasome has been shown to trigger parkinsonian neurodegeneration (McNaught and Jenner 2001; Hardy et al., 2001).
ALP involves the engulfment of cytoplasmic materials into autophagosomes, which are degraded by lysosomal enzymes after fusion of autophagosomes with lysosomes (Kuma et al., 2004) or direct import of proteins into lysosomes (Cuervo, 2004; Mizushima et al., 2008). Autophagy also plays an essential role for the removal of damaged organelles, such as mitochondria. Both, excessive autophagy or reduced autophagic flux can compromise cell survival (Rothermel and Hill, 2007), and several genetic forms of PD are linked to the autophagy-related genes Pink1, Parkin or Uchl1. Autophagy enables cell survival during mitochondrial stress by clearing the damaged organelles (Lee et al., 2012).
One of the main aggregated proteins found to accumulate in nigrostriatal cells during Parkinson's disease is α-synuclein. Aggregation of α-synuclein can obstruct normal cellular transport, leading to impaired intracellular trafficking and/or trapping of cellular organelles in inappropriate locations, this resulting in synaptic and cell dysfunctions (Bartels et al., 2011) (Bellucci A., et al., 2012; Cookson MR., 2005; Games D., et al., 2013; Hunn BH., et al., 2015). Importantly, accumulation of α-synuclein affects mitochondrial trafficking. The polarity and correct function of different types of cells depend on an efficient transport of mitochondria to areas of high energy consumption (Sheng, 2014). Therefore, the correct distribution of mitochondria to various parts of a cell is essential to preserve cell function (Schwarz, 2013; Zhu et al., 2012).
How It Is Measured or Detected
1. Evaluation of UPS function. General turnover assays: quantitative evaluation can be based on the detection of increased ubiquitin or ubiquinated proteins, as well as proteasomal subunits, either by immunocyto/histochemistry or by western blotting (Rideout et al., 2001; Ortega and Lucas, 2014). UPS activity can be continuously monitored by quantitating (by mean of flow cytometry or microscopy) the level of e.g. EGFP-degron fusion proteins (green fluorescent protein) that are selectively degraded by the proteasome (Bence et al., 2001).
Proteasome activity assay. Various fluorogenic substrates (e.g., Suc-Leu-Leu-Val-Tyr-AMC for the chymotrypsin-like activity) can be used for the determination of proteasomal activity in in vivo or in vitro applications. These substrates may be applied to tissue or cell homogenates, but specific measurements require partial purification of the proteasome (Kisselev and Goldberg, 2005).
Detection of α-synuclein (AS) aggregates. The most common methods to detect AS aggregates use immunostaining for AS (in cells or in tissues). In cell culture, AS may also be epitope-tagged or coupled to GFP to allow an indirect detection. The detection of small, not microscopically-visible AS aggregates is indicative of protease-resistance. Tissue slices may be exposed to proteases before immunostaining for AS. Alternatively, small or large aggregates may be biochemically enriched by differential centrifugation and proteolytic treatment, and then analyzed, e.g., by western blot, mass spectrometry or ELISA-like immunoquantification.
2. Evaluation of ALP function.
Quantification of lysosomes or autophagosomes. Disturbances of ALP often result in counter-regulations that can be visualized by staining of lysosomes or parts of the autophagy system. Several weakly basic dyes can be used to stain acidic organelles (lysosomes) in live cells. For example, the dye LysoTracker Red stains lysosomes and can be used to monitor autophagy (Klionsky et al., 2007; Klionsky et al., 2008). The autofluorescent drug monodansylcadaverine (MDC) has also been used as autophago-lysosome marker (Munafó and Colombo, 2002). A convenient way to stain lysosomes in tissue or fixed cells is the use of antibodies against the Lysosomal-Associated Membrane Protein 1 (LAMP-1) (Rajapakshe et al., 2015) or against cathepsins (Foghsgaard et al., 2001). For qualitative or semiquantitative estimates of lysosomes and related organelles, transmission electron microscopy has been frequently used (Barth et al., 2010).
Monitoring of autophagy-related molecules. The amount and the localization of autophagy-related proteins can change during disturbance of the ALP. Especially in cell culture, but also in transgenic mice, various techniques have been used to monitor autophagy by mean of fluorescence-tags or other substrates, e.g., ATG, autophagy-related protein or autophagy substrates, to monitor their fate in cells and thus provide information on disturbed ALP, or the over-expression of GFP–LC3, in which GFP (green fluorescent protein) is expressed as a fusion protein at the amino terminus of LC3 (microtubule-associated protein 1A/1B-light chain 3), which is the a mammalian homologue of S. cerevisiae ATG8 (Kadowaki and Karim, 2009).
Monitoring autophagic flux. The lysosomal degradation of the autophagic cargo constitutes the autophagic flux, which can be measured by assessing the rate of turnover of long-lived proteins that are normally turned over by autophagy (Bauvy et al., 2009). This is performed by labelling intracellular proteins with either [14C]-leucine or [14C]-valine, followed by a long culture period in standard medium. The release of radioactive leucin or valin into the culture medium corresponds to the protein degradation rate in cells, and it may be measured by liquid scintillation counting.
Monitoring the conversion of LC3-I to LC3-II. The progression of autophagy (autophagic flux) can be studied by the conversion of LC3-I into LC3-II (i.e. a post-translational modification specific for autophagy) by mean of Western blot analysis. The amount of LC3-II correlates with the number of autophagosomes. Conversion of LC3 can be used to examine autophagic activity in the presence or absence of lysosomal activity (Klionsky et al., 2007; Klionsky et al., 2008). The technology can also be used in vivo, e.g. by the use of transgenic mice that overexpress GFP–LC3 (Kuma et al., 2004).
3. Evaluation of intracellular transport of mitochondria and other organelles.
A range of technologies has been used to visualize mitochondrial dynamics in live cells (Jakobs, 2006; Grafstein and Forman, 1980). They usually employ a combination of mitochondrial labelling with fluorescent dyes (e.g. DiOC6 (3, 3′-Dihexyloxacarbocyanine iodide), JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′ tetraethylbenzimida-zolylcarbo-cyanine iodide), MitoTracker, MitoFluor probes, etc.), followed by video- or confocal microscopy for live cell imaging (Schwarz, 2013; Pool et al., 2006). Most frequently, mitochondrial mobility is observed along neurites, and measurable endpoints may be mitochondrial speed and direction with regard to the cell soma (Schildknecht et al. 2013). Additionally, also mitochondrial fusion and fission have been monitored by such methods (Exner et al., 2012). The transport of other organelles along neurites may be monitored using similar methods, and the microtubule structures that serve as transport scaffold may be co-stained.
Domain of Applicability
The ubiquitin proteasome system is highly conserved in eukaryotes, from yeast to human. Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues of eukaryotic organisms. For instance, drosophila has been used as PD model to study the role of ubiquitin in α-synuclein induced-toxicity (Lee et al., 2009). Human and yeast ubiquitin share 96% sequence identity. Neither ubiquitin nor the ubiquitination machinery are known to exist in prokaryotes. Autophagy is ubiquitous in eukaryotic cells and is the major mechanism involved in the clearance of oxidatively or otherwise damaged/worn-out macromolecules and organelles (Esteves et al., 2011). Due to the high degree of conservation, most of the knowledge on autophagy proteins in vertebrates is derived from studies in yeast (Klionsky et al., 2007). Autophagy is seen in all eukaryotic systems, including fungi, plants, slime mold, nematodes, fruit flies and insects, rodents (i.e., laboratory mice and rats), and humans. It is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation.
Evidence for Perturbation by Stressor
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