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Event Title

Proteostasis, impaired

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Inhibition of the mitochondrial complex I of nigra-striatal neurons leads to parkinsonian motor deficits KE Moderate

Taxonomic Applicability

Name Scientific Name Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Moderate NCBI

Level of Biological Organization

Biological Organization

How this Key Event works

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

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

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 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.

Evidence Supporting Taxonomic 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.


Barth S., Danielle Glick, and Kay F Macleod, Autophagy: assays and artifacts. J Pathol. 2010 Jun; 221(2): 117–124.

Bartels T, Choi JG, Selkoe DJ (Sep 2011). "α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation". Nature 477 (7362): 107–10.

Bauvy C, Meijer AJ, Codogno P. Assaying of autophagic protein degradation. Methods Enzymol. 2009;452:47–61.

Bellucci A., M. Zaltieri, L. Navarria, J. Grigoletto, C. Missale, and P. Spano, “From 𝛼-synuclein to synaptic dysfunctions: new insights into the pathophysiology of Parkinson’s disease,” Brain Research, vol. 1476, pp. 183–202, 2012.

Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin–proteasome system by protein aggregation. Science 2001;292:1552–5.

Ciechanover A. (1998) The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17, 7151±7160.

Ciechanover A., and Brundin P., 2003, The Ubiquitin Proteasome System in Neurodegenerative Diseases: Sometimes the Chicken, Sometimes the Egg. Neuron, 427–446

Cookson MR., “The biochemistry of Parkinson’s disease,” Annual Review of Biochemistry, vol. 74, pp. 29–52, 2005.

Cuervo A.M., “Autophagy: many paths to the same end,” Molecular and Cellular Biochemistry, vol. 263, no. 1, pp. 55–72, 2004.

Exner N, Lutz AK, Haass C, Winklhofer KF. Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences. EMBO J. 2012 Jun 26;31(14):3038-62.

Esteves AR, Arduíno DM, Silva DF, Oliveira CR, Cardoso SM. 2011. Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD. Parkinsons Dis. 2011:693761.

Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M, Jäättelä M. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol. 2001 May 28;153(5):999-1010.

Games D., P. Seubert, E. Rockenstein et al., “Axonopathy in an 𝛼-synuclein transgenic model of Lewy body disease is associated with extensive accumulation of c-terminal-truncated𝛼-synuclein,” American Journal of Pathology, vol. 182, no. 3, pp. 940–953, 2013.

Grafstein B., and Forman DS. Intracellular transport in neurons. Physiological Reviews Published 1 October 1980 Vol. 60 no. 4.

Hardy J. Rideout, Kristin E. Larsen, David Sulzer and Leonidas Stefanis, Proteasomal inhibition leads to formation of ubiquitin/a-synuclein-immunoreactive inclusions in PC12 cells. Journal of Neurochemistry, 2001, 78, 899±908

Hunn BH., S. J. Cragg, J. P. Bolam, M. G. Spillantini, and R. Wade-Martins, “Impaired intracellular trafficking defines early Parkinson’s disease,” Trends in Neurosciences, vol. 38, no. 3, pp.178–188, 2015.

Ii K., Ito H., Tanaka K. and Hirano A. (1997) Immunocytochemical co-localization of the proteasome in ubiquitinated structures in neurodegenerative diseases and the elderly. J. Neuropathol. Exp. Neurol. 56, 125-131.

Jakobs S., High resolution imaging of live mitochondria, 2006, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1763, Issues 5–6 Pages 561–575

Kadowaki M, Karim MR. Cytosolic LC3 ratio as a quantitative index of macroautophagy. Methods Enzymol. 2009;452:199–213. [PubMed]

Kisselev AF, Goldberg AL. Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol. 2005;398:364–378.

Klionsky DJ., Ana Maria Cuervo & Per O. Seglen. Methods for Monitoring Autophagy from Yeast to Human. Autophagy 2007, 3:3, 181-206; Klionsky D.J., Abeliovich H., Agostinis P., Agrawal D.K., Aliev G., Askew D.S., Baba M., Baehrecke E.H., Bahr B.A., Ballabio A., et al Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008;4:151-175.

Korolchuk VI, Menzies FM, Rubinsztein DC (2010) Mechanisms of cross-talk between the ubiquitin–proteasome and autophagy–lysosome systems. FEBS Lett 584:1393–1398

Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. J. Molecular cell 40:280–293.

Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032–1036.

Lee J, Giordano S, Zhang J; Giordano; Zhang (January 2012). "Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling". Biochem. J. 441 (2): 523–40.

Lee FK, Wong AK, Lee YW, Wan OW, Chan HY, Chung KK. The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease. J Neurochem. 2009 Jul;110(1):208-19

McNaught K. S. and Jenner P. (2001) Proteasomal function is impaired in substantia nigra in Parkinson's disease. Neurosci. Lett. 297, 191-194.

Mizushima N. et al., 2008. Autophagy fights disease through cellular self-digestion. Nature. 451(7182):1069-75. Review.

Munafó DB, Colombo MI. Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24. Traffic. 2002 Jul;3(7):472-82.

Ortega Z. and Lucas J.J. (2014) Ubiquitin–proteasome system involvement in Huntington’s disease Front Mol Neurosci. 2014; 7: 77.

Pool M., Rippstein P., Mcbride H. Kothary R., 2006 Trafficking of Macromolecules and Organelles in Cultured Dystonia musculorum Sensory Neurons Is Normal. J. Comparative Neurology 494:549–558 (2006)

Rajapakshe AR, Podyma-Inoue KA, Terasawa K, Hasegawa K, Namba T, Kumei Y, Yanagishita M, Hara-Yokoyama M. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells. Exp Cell Res. 2015 Feb 1;331(1):211-22. doi: 10.1016/j.yexcr.2014.09.014.

Ravikumar B, Sarkar S, Davies JE et al (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383–1435. doi:10.1152/physrev.00030.2009

Rothermel BA, Hill JA (2007) Myocyte autophagy in heart disease: friend or foe? Autophagy 3:632–634.

Rideout HJ, Larsen KE, Sulzer D, Stefanis L. 2001.Proteasomal inhibition leads to formation of ubiquitin/a-synuclein-immunoreactive inclusions in PC12 cells. Journal of Neurochemistry. 78, 899-908.

Schildknecht S, Karreman C, Pöltl D, Efrémova L, Kullmann C, Gutbier S, Krug A, Scholz D, Gerding HR, Leist M. Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases. ALTEX. 2013;30(4):427-44.

Schwarz TL.Mitochondrial trafficking in neurons. Cold Spring Harb Perspect Biol. 2013 Jun 1;5(6). pii: a011304.

Sheng ZH., Mitochondrial trafficking and anchoring in neurons: new insight and implications. J of Cell Biology, vol. 204. No.7 pp. 1087-1098, 2014.

Spillantini M. G., Schmidt M. L., Lee V. M., Trojanowski J. Q., Jakes R. and Goedert M. (1997) Alpha-synuclein in Lewy bodies.Nature 388, 839-840.

Sulzer D. and Zecca L. (2000) Intraneuronal dopamine-quinonem synthesis: a review. Neurotoxicity Res. 1, 181-195.

Zhu XH, Qiao H, Du F, Xiong Q, Liu X, Zhang X, Ugurbil K, Chen W. Quantitative imaging of energy expenditure in human brain. Neuroimage. 2012;60(4):2107-17)