N/A, Mitochondrial dysfunction 1 leads to Impaired, Proteostasis

In any cell type, including neurons, the protein homeostasis (proteostasis) plays a key role in cellular functions. There are two major systems involved in the removal of damaged cellular structures (e.g. defective mitochondria) and misfolded or damaged proteins, the ubiquitin-proteasome system (UPS) and the autophagy–lysosome pathway (ALP). These processes are highly energy demanding and highly susceptible to oxidative stress. Upon mitochondrial dysfunction UPS and ALP functions are compromised resulting in increased protein aggregation and impaired intracellular protein/organelles transport (e.g. Zaltieri et al., 2015; Song and Cortopassi, 2015; Fujita et al., 2014; Esteves et al., 2011; Sherer et al., 2002).

As described in the studies above (Empirical support for linkage) a quantitative or semi-quantitative relationship has been established between rotenone-induced mitochondrial dysfunction and the impairment of UPS/ALP function. Below some representative studies are reported as examples for how such quantitative evaluations can be performed.

• Human neuroblastoma SK-N-MC or human embryonic kidney (HEK) cells were exposed to rotenone at 100 nM for 24 or 48 hrs (for further details see Chou et al., 2010).

• PD patient-derived fibroblasts (vs Ctr fibroblasts) treated with rotenone (20 and 500 μM for 6 h for the evaluation of protein quality control system or 100 nM, 1 μM and 10 μM for 1 h for redox experiments) showed reduction of UPS function (as shown by higher induction of 20S proteasome activity in PD fibroblasts vs Ctr after both 20 and 500 μM rotenone administration). An increase of LC3-II accumulation in both groups (PD and Ctr) after exposure to 500 μM rotenone was observed suggesting that (Ambrosi et al. 2014).

• Human neuroblastoma cells (SK-N-MC) after short treatment with rotenone (1 week) elevated soluble α-synuclein protein (41 ± 16% increase) levels without changing mRNA levels, suggesting impairment of α-synuclein degradation via UPS. Chronic rotenone exposure (4 weeks) increased levels of insoluble α-synuclein (29 ± 9% increase) and ubiquitin (87 ± 14% increase) (Sherer et al., 2012).

• SHSY-5Y cells treated with rotenone (500 nM, 24 h) showed a ~2 fold increase in DCF fluorescence compared to untreated cells (indicative of intracellular ROS). Additionally, rotenone elevated cytosolic calcium (about 35-40% increase vs Ctr), ER-stress (about 45% increase vs Ctr), impaired UPS function (~3 fold increase of insoluble protein aggregate vs Ctr). Inhibition of Rac1 (Rho-like GTPase) mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function (Pal et al. 2014).

• Human neuronal SH-SY5Y cells treated with rotenone (10 μM, for 24 hr showed accumulation of high molecular weight ubiquitinated bands (by immunoblotting – qualitative - assay), and increase of both mitochondrial- (~5 fold increase vs Ctr) and cytosolic- cytochrome c fractions (~1.2 fold increase vs Ctr). Rapamycin pre-treatment (3 μM, for 48 hr prior addition of rotenone) diminished rotenone-induced effects, as shown by enhanced degradation of ubiquitinated proteins, and reduced levels of cytosolic cytochrome c. Also, rapamycin promoted mitophagy (as shown by lysosome and mitochondria co-localization within the cells) (Pan et al. 2009).

Examples of quantitative evaluation of this KER

Fig.1. Dose and time dependent striatal proteasome activity after MPTP continuously infused upto 28 days measured by relative chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolysing (PGPH) proteasome activities in mice. Delayed and prolonged inhibition of proteasome activity after continuous MPTP administration (1, 5, or 30 mg/kg MPTP daily) for the indicated time periods. Asterisks indicate statistically significant differences (P _<0.05) from baseline proteasome activity (single asterisk) or from both baseline proteasome activity and activity after lower MPTP doses (1 and 5 mg/kg, daily, double asterisk; n =5 mice) (Fornai et al., 2005, Fig. 2 B).

Fig. 2. Effect of α-synuclein deletion on MPTP toxicity. Proteasome activity in control and alpha-synuclein KO mice continuously infused for 28 days with MPTP (30 mg/kg of body weight daily, striatum concentration approximately 13 uM). Proteasome activities in the substantia nigra are depicted as percent of control (means +/- SEMs) as a function of time after beginning of the infusions (five mice per group). Asterisks indicate statistically significantly different values (P < 0.05) from controls (Fornai et al., 2005).

Fig. 3. α-Synuclein levels were selectively increased in the ventral midbrain (VMB) region of rotenone-infused rats with or without lesion. α-Synuclein levels, as determined from Western blot analysis, from rotenone-treated rats were expressed as a percentage of values from control vehicle-infused rats. Results are mean ± SEM (n = 3 control, 6 rotenone with lesion, 3 rotenone with no lesion) *P < 0.05 vs. vehicle-infused rats (from Betarbet et al., 2006, Fig. 3A).

Fig. 4. Bar graph showing the effects of rotenone and lactacystin on α-synuclein levels after 4 weeks of rotenone exposure (5 nM) in vitro, on SK-N-MC human neuroblastoma cells. Rotenone alone increased α-synuclein levels, but lactacystin alone did not. α-Tocopherol attenuated the rotenone-induced increase in α-synuclein. Results are mean ± SEM (n = 4). *P < 0.05 vs. solvent-treated cells. CC, control cells; RC, rotenone-treated cells; C-Lac or CL, lactacystin treated cells; R-lac or RL, rotenone and lactacystin treated cells; R-AT, rotenone and α-tocopherol treated cells (from Betarbet et al., 2006, Fig. 5B).

Fig.5. Levels of ubiquitinated proteins were estimated in solubilized protein fractions from SK-N-MC cells collected at the end of each week of rotenone treatment (5 nM), using gel electrophoresis and immunoblotting. Quantitative analysis demonstrated significant increases in ubiquitinated protein levels 4 weeks after rotenone treatment and after proteasomal inhibition with lactacystin. Band intensities were expressed as % of control. Results represent mean ± SEM. *P < 0.05 compared to control (from Betarbet et al., 2006, Fig. 8C).

Fig. 6. Effects of rotenone on the activity of proteasome. Proteasome activity in the cytoplasmic fraction of cells treated with 25 nM (A) or 50 nM (B) rotenone was measured fluorometrically in the absence (open triangles and circles) or presence (solid triangles and circles) of exogenously added ATP (2 mM) (from Shamoto-Nagai et al., 2003, Fig. 6).

Table. 1. These studies showed that rotenone caused a reduction in UPS activity (measured by 26S and 20S proteasome activity) in a dose-dependent manner. Further studies showed that rotenone increases proteasome subunit degradation, but does not alter synthesis (Western blot and RT-PCR studies, reviewed in Chou et al., 2010). Dose- and time- dependent striatal proteasome activity is also shown after MPTP continuously infused up to 28 days measured by relative chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolysing (PGPH) proteasome activities in mice (Fornai et al. 2005).

• PD patient-derived fibroblasts (vs Ctr fibroblasts) showed reduction of UPS function (by ~33%) and higher accumulation of ubiquitinated proteins (by ~2 fold) in PD as compared to control fibroblasts at baseline. Treatment with rotenone (20, 500 μM, 6hr) caused a higher induction of 20S proteasome activity in PD fibroblasts vs Ctr. An increase of LC3-II accumulation (indicative of autophagic vesicle accumulation) in both groups (PD and Ctr) after exposure to 500 μM rotenone was observed (Ambrosi et al. 2014).

• Human neuroblastoma cells (SK-N-MC) after short treatment with rotenone (1 week) elevated soluble α-synuclein protein (41 ± 16% increase) levels without changing mRNA levels, suggesting impairment of α-synuclein degradation via UPS. Chronic rotenone exposure (4 weeks) increased levels of insoluble α-synuclein (29 ± 9% increase) and ubiquitin (87 ± 14% increase) (Sherer et al., 2012).

• SHSY-5Y cells treated with rotenone (500 nM, 24 h) showed a ~2 fold increase in DCF fluorescence compared to untreated cells (indicative of intracellular ROS). Additionally, rotenone elevated cytosolic calcium (about 35-40% increase vs Ctr), ER-stress (about 45% increase vs Ctr), impaired UPS function (~3 fold increase of insoluble protein aggregate vs Ctr). Inhibition of Rac1 (Rho-like GTPase) mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function (Pal et al. 2014).

• Human neuronal SH-SY5Y cells treated with rotenone (10 μM, for 24 hr showed accumulation of high molecular weight ubiquitinated bands (by immunoblotting – qualitative - assay), and increase of both mitochondrial- (~5 fold increase vs Ctr) and cytosolic- cytochrome c fractions (~1.2 fold increase vs Ctr). Rapamycin pre-treatment (3 μM, for 48 hr prior addition of rotenone) diminished rotenone-induced effects, as shown by enhanced degradation of ubiquitinated proteins, and reduced levels of cytosolic cytochrome c. Also, rapamycin promoted mitophagy (as shown by lysosome and mitochondria co-localization within the cells) (Pan et al. 2009).

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.