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

The biological relationship between Mitochondrial dysfunction and Impaired proteostasis (unbalanced protein homeostasis) that involves dysregulation of proteins degradation (misfolded or damaged) as well as removal of cell organelles is partly understood. Under physiological conditions, mechanisms by which proteostasis is ensured include regulated protein translation, chaperone assisted protein folding and functional protein degradation pathways. Under oxidative stress, the proteostasis function becomes burdened with proteins modified by ROS (Powers et al., 2009; Zaltieri et al., 2015). These changed proteins can lead to further misfolding and aggregation of proteins (especially in non-dividing cells, like neurons). Particularly in DA cells, oxidative stress from dopamine metabolism and dopamine auto-oxidation may selectively increase their vulnerability to CI inhibitors (such as rotenone) and cause additional deregulation of protein degradation (Lotharius and Brundin, 2002; Esteves et al., 2011). As most oxidized proteins get degraded by UPS and ALP (McNaught and Jenner, 2001), mitochondrial dysfunction and subsequent deregulation of proteostasis play a pivotal role in the pathogenesis of PD (Dagda et al., 2013; Pan et al., 2008; Fornai et al., 2005; Sherer et al., 2002). It is also well documented that increased oxidative stress changes the protein degradation machinery and leads to a reduction of proteasome activity (Lin and Beal, 2006; Schapira, 2006).

Based on the existing in vitro and in vivo data it is suggested that mitochondrial dysfunction impairs protein homeostasis through oxidative and nitrosative stress resulting in protein aggregation, disruption of microtubule assembly and damaged intracellular transport of proteins and cell organelles.

Mitochondrial dysfunction by rotenone or MPP⁺ reduces UPS activity:

• Mitochondrial dysfunction induced by systemic and chronic CI inhibition by rotenone, results in a selective inhibition of proteasomal function in the midbrain (not in cortical or striatal homogenates) of rats that had lost the TH-positive terminals in the striatum. Initially, proteasomal activity showed an acute increase prior to a decrease by 16-31 %, during chronic rotenone exposure (3.0 mg/kg/day, through subcutaneous osmotic pump during 5 weeks). In the same animals a significant and selective increase in ubiquitinated proteins was observed (~ 25%) in the ventral midbrain of lesioned rats, indicating an increase in the proteins levels that have been marked for degradation by UPS. These results were confirmed immunocyto-chemically, pointing out that ubiquitin levels were elevated selectively in DA neurons present in SNpc (Betarbet et al., 2006).

• Nigral neurons in chronically rotenone-treated rats (up to 5 weeks, continuous intrajugular infusion of rotenone at 2.5 mg/kg/day) accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and α-synuclein (the main protein of Lewy bodies observed in PD) (qualitative data, obtained by immuno-electron microscopy) (Betarbet et al., 2000).
• Inhibition of proteasomal function was also observed in in vitro systems using SK-N-MC human neuroblastoma. Exposure to 5 nM rotenone, for up to 4 weeks caused 60% increase in the levels of ubiquinated proteins, suggesting that chronic exposure to rotenone increased the level of misfolded or oxidized proteins targeted for degradation by UPS (Betarbet et al., 2006).

• To determine whether rotenone-induced proteasomal inhibition was due to CI inhibition or direct effects of rotenone on the UPS, proteasomal activity was determined in SKN-MC cells expressing the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which acts as a "replacement" for the entire CI in mammalian cells (Bai et al., 2001; Seo et al., 2000, 2002). The obtained results confirmed that rotenone-induced proteasomal dysfunction is due to CI inhibition and not to direct effects of rotenone on proteasomal function (Betarbet et al., 2006). In the same study the decreased proteasomal activity and an accumulation of ubiquitinated proteins was completely prevented by continuous treatment with α-tocopherol (62.5 μM added 1 week prior to and continuously thereafter along with 5 nM rotenone) (qualitative data), confirming that oxidative damage played a major role in rotenone-induced proteasomal dysfunction rather than bioenergetic defects. Indeed, chronic, low levels of rotenone exposure did not changed significantly ATP levels (111.5 ± 1.5% of control), but produced ROS (not shown in this study). Similar results were published by Shamoto-Nagai's group (Shamoto-Nagai et al. 2003).

• Rotenone significantly lowered UPS activity in a concentration dependent manner in HEK (human embryonic kidney cells) and SK-N-MC human neuroblatoma cells even after 24 h exposure to doses as low as 10 nM. It caused a reduction in the 20S proteasome activity (by 5-25%) and of the 20S proteasome subunit (by 20-60%) (as shown by increase of GFP-U fluorescence) (Chou et al., 2010). Similar results were obtained using other pesticides that inhibit CI, including pyridaben and fenazaquin (Wang et al., 2006). This effect was mediated by oxidative stress as anti-oxidants, such as butylated-hydroxy toluene (BHT), and catalase attenuated rotenone-induced UPS inhibition. Additionally, nitric oxide (NO) and peroxinitrite contributed to this effect as well, since neuronal nitric oxide synthase (nNOS) inhibitor (LNMMA) attenuated rotenone-induced proteasome inhibition by 20% (Chou et al., 2010) indicating that both oxidative and nitrative stress can directly inhibit the proteasome activity through increased degradation of proteasome subunits. The same mechanisms of proteasome inhibition were suggested by many other studies (e.g. Szweda et al, 2002; Osna et al., 2004; Shamoto-Nagai et al., 2003).

• CI inhibition-induced proteasomal dysfunction has been reported in ventral mesencephalic cultures following acute rotenone or MPP⁺ exposure (Hoglinger et al., 2003). In DA neurones derived from rat (embryonic day 15.5) ventral mesencephalon, it has been showen that proteasome inhibition (by 100 nm epoxomicin) exacerbated the neurotoxicity of CI inhibitors (by mean of rotenone 30 nM, or MPP⁺ 3 µM, for 24 hr). All three proteasomal peptidase activities (i.e., chymotrypsin (CT)-like, trypsin (T)-like, and peptidylglutamyl-peptide hydrolase (PGPH) activity) significantly decreased in cultures upon 6 hr treatment with 30 nM rotenone (by 50+-60%) or 30 µM MPP⁺ (by 25-30%) (Hoglinger et al., 2003).

• CI inhibition-induced proteasomal dysfunction has been reported in human SH-SY5Y neuroblastoma cells following acute rotenone exposure (Shamoto-Nagai et al., 2003). After 96 hr of incubation with 25 or 50 nM rotenone, the activity was reduced respectively to 28.7% and 21.9% of control, and adding ATP did not increase the activity. After 120 hr, the activity was virtually undetectable (with or without added ATP). On the contrary, the levels of the proteins composing proteasome did not change with rotenone treatment (Shamoto-Nagai et al., 2003).

• The ability of rotenone to cause proteasome inhibition via disruption of microtubules (MT) assembly has been also documented. In human embryonic kidney (HEK) and neuroblastoma SK-N-MC cells rotenone (10-100-100 nM, 24 hr) was found to inhibit 26S UPS activity (by 25%, at 10 nM) (Chou et al., 2010). Rotenone was found to interfere with MT assembly at concentrations as low as 10 nM, providing evidence that there could be additional mechanisms implicated in the rotenone induced UPS inhibition, possibly mediated by nitric oxide (NO). In the same study, nocodazole, a MT disrupter (positive control), strongly inhibited the UPS activity (e.g., 10 µM nocodazole caused ~80% decrease of 26S UPS activity) (Chou et al., 2010).

• Oxidative stress triggered by the MPP⁺ inhibited CI (1 mM, for 2-6-24 hr) led to a decrease in proteolytic activity, as shown in NT2 human teratocarcinoma cells containing mitochondrial DNA (ρ+) and NT2 cells depleted of mtDNA (ρ0) (Domingues et al., 2008). In particular, MPP⁺ (1 mM, 2 hr) elevated ubiquitinylated protein content (by ~3 fold compared to untreated Ctr), and after 24 hr induced a significant decrease of chymotrypsin-like activity (by ~30%) and peptidyl-glutamyl peptide hydrolytic-like activity (by ~75%) compared to untreated cells (Domingues et al., 2008).

• Mice following continuous MPTP infusion (1-5-30 mg/kg daily) exhibited inhibition of the UPS (respectively by 40-50-60%) and increased inclusions of ubiquitin and α-synuclein in the neurons in the substantia nigra (Fornai et al., 2005).

• A mouse model of mitochondrial CI deficiency (Ndufs4-/- mice) showed an impaired 20S proteasomal activity (by ~50%), leading to increased ubiquitin protein levels (by ~40%) in the substantia nigra (not in cortex and hippocampus), increased of ubiquitin+/TH+ neurons (by ~2 fold, compared to WT mice), and increased ubiquitinated neurofilaments in the midbrain (values of 1.2 - 2.8 vs 1.0 in WT) (Song and Cortopassi, 2015).

Human studies.

• PD patients appear to have an impaired UPS. The presence of aggregated, poly-ubiquitinated proteins in Lewy Bodies indicates that proteolytic dysfunction and proteo-toxicity are critical steps in the pathogenic cascade of PD (Betarbet et al., 2005). In this regard, impairment of proteasomal activity and reduced expression of proteasomal subunits have been reported selectively in subtantia nigra of sporadic PD post-mortem brains (McNaught et al., 2003; McNaught and Jenner, 2001). In particular, in PD, there was a 40.2% reduction in the amount of α-subunits in the SNc. On the opposite α-subunits levels were increased by 9.2% in the cerebral cortex and by 29.1% in the striatum in PD compared to Ctr (McNaught et al., 2003). Chymotrypsin-like, trypsin-like, and peptidyl glutamyl-peptide hydrolytic (PGPH) 20/26S proteasomal activities were significantly decreased in the substantia nigra (by 43.9%, 45.9%, and 44.6% respectively) (not in the cortex or striatum) in PD patients. At the same time, in PD there was a marked increase in the levels of PA700 subunits (the 19S regulatory complex of the 26S proteasome) in the frontal cortex and/or the striatum compared to controls, while in the SNpc PA700 subunits resulted decreased up 33%, whereas levels of nigral PA28 were almost undetectable in both normal and PD subjects (McNaught et al., 2003).

• Steady-state levels of soluble AF-6 (modulates parkin ubiquitin-ligase activity) have been found significantly lower in the caudate/putamen (~66% lower) as well as in the SN of PD patients (~66% lower). AF-6 was also detected in ∼25% of mature Lewy bodies and in occasional Lewy neurites in the substantia nigra of the four PD brains analysed, and may contribute to the disruption of mitochondrial homeostasis (Haskin et al. 2013).

• HDAC6 has recently been identified by immunocytochemistry as a constituent in Lewy bodies of PD and dementia with LBs (DLB), as well as in glial cytoplasmic inclusions in multiple system atrophy (MSA) (Kawaguchi et al. 2003; Miki et al. 2011; Chiba et al. 2012). HDAC6 is considered a sensor of proteasomal inhibition and a cellular stress surveillance factor. Upon proteasomal inhibition, HDAC6 is relocated and recruited to polyubiquitin-positive aggresomes. HDAC6 inhibition elicits tubulin acetylation and restores microtubule (MT)-dependent transport mechanisms in neurons (Richter-Landsberg and Leyk, 2013).

• Basal activity of 20S proteasome was significantly reduced (by ~33%) in PD as compared to control fibroblasts. Higher accumulation of ubiquitinated proteins (by ~2 fold), representative of impaired 26S proteasome function, were found in PD as compared to Ctr cells at baseline. In the presence of rotenone (20 and 500 μM, 6 hr) PD-derived fibroblasts showed a higher induction of 20S proteasome activity (~15% higher) as compared to Ctr fibroblasts, with no significant changes in autophagy (except from increased LC3-II accumulation in both groups after exposure to 500 μM rotenone) (Ambrosi et al., 2014).

Mitochondrial dysfunction by rotenone or MPP⁺ deregulates ALP activity

• Exposure to rotenone (10 μM, 24 hr) induced neurotoxicity in human neuronal SH-SY5Y cells (number of dead cells was 8 folds higher than Ctr group) and pre-treatment with rapamycin (3 μM, 48 hrs) (strong inducers of autophagy) robustly protected against rotenone-mediated toxicity (number of dead cells was 3 folds higher than Ctr group) and this was due to the induction of autophagy. Indeed, suppression of autophagy (by silencing of Atg5) blocked the neuroprotection of rapamycin (Pan et al., 2009).

• Similar results were produced using kaempferol (6 μM, 1 hr prior addition of rotenone) and rotenone (50 nM, max up to 24 hr) on SH-SY5Y cells. Kaempferol was found to counteract rotenone-induced effects (see KER2) and these protective effects were related to induction of autophagy (6 hr kaempferol induced LC3-II formation, as shown by Western blot) (Filomeni et al., 2012).

• Treatment of SH-SY5Y cells with high doses of rotenone (500 nM, 48 hr) induced Atg5–Atg12 dependent autophagy, which leads to lysosomal dysfunction, increased p62 levels, and an aberrant accumulation of α-synuclein (Pan et al., 2009; Dadakhujaev et al., 2010). In particular, in α-synuclein expressing SH-SY5Y cells Atg5–Atg12 were increased by addition of rotenone and rapamycin (100 nM, 48 hr). Co-treatment with rotenone and autophagy inhibitors (e.g., 3-MA, bafilomycin or wortmannin) similarly diminished the level of Atg5–Atg12 in α-synuclein expressing cells (western blot analyses) (Dadakhujaev et al., 2010).

• A few studies have suggested that rotenone can act as an inducer of autophagic flux. For instance, treating human embryonic kidney cells (HEK 293) and U87 glioma cells with rotenone (50 μM, for 0-72 hr) caused cell death (in HEK 293 cells, rotenone induced 30% cell death, after 72 hr; in U87 cells, 40%) by upregulating autophagy and mitophagy (as shown by increase of cells with AVOs (indicative of autophagosomes and autolysosomes, analysed by flow cytometry): by ~14% in HEK 293 cells, and by ~20% in U87 cells, as compared to untreated cells, 0%), a process that is supposed to be triggered by mitochondrial superoxide (Chen et al., 2007).

• Increased autophagic flux has been observed in SH-SY5Y cells and primary cortical neurons treated respectively with 1 μM and 250 nM of rotenone. Rotenone elicited increases in autophagy (~ 2 folds vs Ctr) and mitophagy (i.e., as shown by the percentage of GFP-LC3 puncta colocalizing with mitochondria (~ 4 folds vs Ctr), indicating a preferential increase in “mitophagosomes” relative to total autophagosomes. Additionally, rotenone induced a decrease in p62 (SQSMT1), levels (~40% decrease with 250 nM), consistent with increased autophagic flux. This effect was reversed by co-treating cells with bafilomycin A2, a specific inhibitor of vacuolar-type H(+)-ATPase, or by RNAi (knockdown of ATG7 and ATG8/LC3). The mechanism by which LC3 recognizes damaged mitochondria in rotenone-treated neurons involves, among others, the externalization of cardiolipin and recruitment of LC3 at the mitochondria initiating rotenone induced-mitophagy and lysosomal-mediated degradation of mitochondria (Chu et al., 2013).

• In the study by Wu et al., (2015) chronically rotenone-treated rats (male Lewis rats received rotenone 1mg/kg subcutaneously twice a day for 8 weeks) had a robust loss of TH+ neurons in striatum (~50%) and in SNpc (~30%). However, in the remaining DA neurons of SNpc, cytoplasmic inclusions containing α-synuclein were observed (~7% of α-synuclein+/TH+ cells vs ~2% in Ctr), probably due to rotenone-induced decreased degradation of the autophagosomes (upregulation of LC3-II by ~30%, Beclin 1 by ~10%, and p62 by ~150%, after 24 hr rotenone) indicating decreased ALP function. Compared with the control group, the nigral DA neurons of the rotenone-treated group exhibited an increased diffuse distribution of LAMP2 (~15% vs ~25% Ctr) and cathepsin D (~22% vs ~60% Ctr) instead of punctuate pattern, indicating impaired lysosome integrity and a redistribution of cathepsin D from lysosomes to the cytosol. In parallel in vitro studies by the same group showed that PC12 cells exposed to rotenone (500 nM for 24 hr) underwent increased protein levels (but not mRNA levels) of α-synuclein (~4.5 folds vs Ctr), indicating an impairment of protein degradation. In TEM pictures, the majority of neurons displayed mitochondrial swelling, crista fragmentation, and accumulation of double membrane structures containing damaged mitochondria, which were stalled autophagosomes (Wu et al., 2015).

Similar results, showing impaired autophagic flux resulting in α-synuclein accumulation and the rupture of lysosomes in neuronal cell lines exposed to rotenone have been described in many other studies (e.g. Mader et al., 2012; Sarkar et al., 2014).

• Rotenone produced bidirectional effects on macroautophagy (decrease or increase). This may be attributed to differences in the dosage, the duration, and cell type which can produce variable levels of ROS and mitochondrial damage (Pan et al., 2009; Dadakhujaev et al., 2010; Chen et al., 2007; Filomeni et al., 2012; Mader et al., 2012).

• MPP⁺ (2.5 mM, 24 - 48 hr) increased autophagy (~14 folds increase vs Ctr, of LC3-II) and mitochondrial loss in SH-SY5Y cells (a DA neuronal cell line widely used as a cell culture model of PD) by increased MAP kinase signalling (MEK inhibition by UO126 reversed by both autophagy and mitochondrial loss elicited by MPP+) (Zhu et al., 2007).

• Another study from the same group showed that longer MPP+ treatment (250 μM, 2 weeks) induced formation of enlarged, coarse GFP-LC3 puncta, in a time- and dose-dependent manner (~1.8% of cells presenting coarse GFP-LC3 puncta, vs ~0.2% in Ctr, at 14 days with 250 μM rotenone) (Zhu et al., 2012).

• An in vitro study on MN9D cells (a fusion of embryonic ventral mesencephalic and neuroblastoma cells, used as a model of DA neurons) showed that MPP⁺ (50 μM, for 24 hr) blocked autophagic flux, as evidenced by increased steady-state levels of p62 (qualitative data, Western blot), increased of authophagic vacuoles numbers (~3 folds vs Ctr) along with lysosomal depletion and dysfunction presumably due to leakage of lysosomes, impaired lysosomal biogenesis, and increased proteasomal-mediated degradation of proteins (as shown by time-dependent increase of ubiquitinated proteins, by IC) (Lim et al., 2011).

• In another study human neuroblastoma BE-M17 cells were treated with MPP⁺ (0.25-2.5 mM, 24 hr); Lamp1 protein levels were decreased in a dose-dependent manner in MPP⁺-treated cells (by ~40% at 2.5 mM), without concomitant decreases in mRNA expression levels. Also, LC3-II increased in a dose-dependent manner with MPP⁺ treatment (~3000% increase at 2.5 mM vs Ctr), indicating lysosome depletion and autophagosome accumulation upon MPP⁺ treatment. These data were confirmed in vivo: lysosomal depletion and accumulation of autophagosomes (as shown by ~600% increase of LC3-II, and ~40% decrease of Lamp1, after 1 day of MPTP injection compared to saline) occurred also in MPTP-intoxicated mice (30 mg/kg/day, for 5 consecutive days) (Dehay et al., 2010).

• Other in vivo data support a negative role of MPTP on autophagic flux. Mice were i.p. injected with 2 mg/ml MPTP (30 mg/kg) for 7 days. Suppression of autophagic flux induced by MPTP (~20% reduction vs Ctr) was detrimental to neuronal survival (as shown by ~60% decrease of TH+ neurons). Treating mice with the autophagy inducer rapamycin after seven days of MPTP treatment (daily i.p. injections of 2 mg/ml MPTP (30 mg/kg) for 7 days, followed by 0.1 ml of 20 µg/ml rapamycin by i.v. for an additional 7 days), significantly increased the number of surviving dopamine neurons (~60% TH+ neurons vs ~30% with MPTP alone, as compared to Ctr 100%) and the levels of TH protein (~75% vs ~60% with MPTP alone, as compared to Ctr 100%) and decreased the levels of α-synuclein aggregates (~210% of α-synuclein protein level, vs ~300% with MPTP alone, as compared to Ctr 100%) (Liu et al., 2013).

• Treating mice with the autophagy inducer rapamycin after seven days of MPTP treatment (daily i.p. injections of 2 mg/ml MPTP (30 mg/kg) for 7 days, followed by 0.1 ml of 20 µg/ml rapamycin by i.v. for an additional 7 days), significantly increased the number of surviving dopamine neurons (~75% of TH protein level vs ~60% with MPTP alone) and decreases the levels of α-synuclein aggregates (~210% of α-synuclein protein level, vs ~300% with MPTP alone) (Liu et al., 2013).

MPP+ induced dysregulation of macroautophagy in neurons is discussed in recently published reviews (e.g. Cherra et al., 2010; Jiang et al., 2010). The potential other mechanisms by which rotenone or MPTP induce mitochondrial dysfunction are further discussed in recent publications (e.g. Dagda et al., 2013; Esteves et al., 2011).

Impaired UPS and ALP function leads to α-synuclein aggregation:

α-synuclein is one of the most abundant neuronal proteins (Vekrellis et al., 2011). Several PD-related mutations and environmental toxicants cause autophagy dysfunction and lead to the accumulation of misfolded proteins in DA neurons, including α-synuclein. Both monomeric and aggregated forms of α-synuclein can be degraded by macroautophagy, whereas only wild-type α-synuclein (not Ala30Pro, Ala53Thr and Glu46Lys mutant forms) is degraded by the process of chaperone-mediated autophagy (CMA) (Vekrellis et al., 2011).

• Rotenone-induced α-synuclein aggregation has the ability to inhibit proteasome activity due to its propensity to assemble into filaments (as reviewed in Zaltieri et al., 2015). In particular, expression of α-synuclein was found to inhibit proteasome activity in SH-SY5Y cells. Increased levels of GFP-CL1 band were observed in cells coexpressing GFP-CL1 and α-synuclein (~9000 arbitrary units (au) vs ~500 au in DMSO-Ctr), indicating that proteasome activity is inhibited effectively by expression of α-synuclein (Nonaka and Hasegawa, 2009).

• By using stable PC12 cell lines expressing wild-type (WT) or A53T mutant human α-synuclein it has been shown that cells expressing mutant α-synuclein showed: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins (qualitative data); (2) marked accumulation of autophagic-vesicular structures (qualitative data); (3) reduction of lysosomal hydrolysis and chymotrypsin-like proteasomal function (by ~ 30%, compared to WT) (Stefanis et al., 2001).

• Rotenone- (or MPP+)-induced inhibition of CI results in calcium (Ca2+) release from mitochondria. Calcium rise and oxidative stress cooperatively can promote α-synuclein aggregation (Follett et al., 2013; Goodwin et al., 2013; Nath et al., 2011).
• For instance, to investigate the influence of raised Ca2+ in response to plasma membrane depolarization on the aggregation of a-synuclein, HEK293T and SH-SY5Y neuroblastoma cells have been used and depolarized by addition of KCl to the cell culture medium. After KCl treatment (50 mM) increase of cellular Ca2+ was observed (~90% increase 20 min after KCl treatment), leading to the formation of frequent perinuclear α-synuclein focal aggregates at 26–74 hr post-treatment (qualitative IC images). By adding TMO (a selective T-type Ca2+ channel blocker) no a-synuclein aggregates were detected (Follett et al., 2013).

• Similarly, increased intracellular free Ca2+ (obtained by treating cells with either calcium ionophore or thapsigargin) induced the formation of α-synuclein aggregates in α-synuclein-GFP-transfected 1321N1 glioma cells (~65% increase compared to Ctr-untreated cells) (Nath et al., 2011).

• On the other hand, α-synuclein can control mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions. Silencing of endogenous α-synuclein (siRNA-α-syn) in HeLa cells was found to impair mitochondrial Ca2+ transients (~35% decrease compared to Ctr-scrambled siRNA) and morphology (Calì et al., 2012). Also, α-synuclein oligomerization exacerbates calcium dysregulation by increasing mitochondria permeability transition (Danzer et al., 2007). Therefore, it is possible that mitochondrial dysfunction-induced calcium rise precede the onset of α-synuclein accumulation leading to UPS inhibition (Chou et al., 2010).

• It has been demonstrated that rotenone increased the intracellular calcium levels, triggering aggregation and phosphorylation of α-synuclein in a calcium-dependent manner. The aggregation of α-synuclein in PC12 cells following rotenone exposure was observed in a dose and time-dependent manner (1, 10 and 100 nM for 48 hrs, 3 days, 1 and 3 weeks) (~4 fold increase of α-syn with 100 nM rotenone for 48 hr, vs Ctr; and also, ~2.5 fold increase of α-syn with 1 nM rotenone for 1 week, vs Ctr) as evaluated via a variety of methods, including western blotting, immunofluorescence and electron microscopy. The observed attenuation of autophagy and α-synuclein aggregation was reversed by scavenging calcium (by using the calcium chelator BAPTA at 10 μM). Aggregated α-synuclein is typically degraded by autophagy, but rotenone impaired this process (Yuan et al., 2015).

• Under physiological conditions, α-synuclein is degraded by both the proteasome and autophagy. Mutant α-synuclein inhibits ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway control (e.g. Pan et al., 2009; Betarbet et al., 2000).

• The strongest evidence supporting that mitochondrial dysfunction precedes the onset of α-synuclein pathology derives from studies on rotenone and MPTP in which repetitive exposure of rodents and monkeys to these chemicals via oral, intraperitoneal, intragastric, or nasal administration resulted in the pathological accumulation of α-synuclein in central as well as peripheral neurons (Cannon et al., 2009; Drolet et al., 2009; Mandel et al., 2004; Pan-Montojo et al., 2012 and 2010; Tristão et al., 2014). For example, male Lewis rats were injected with rotenone (2.0 mg/kg, i.p.) and sacrificed at 0, 4, 8, 16, or 32 h after injection and showed α-synuclein and poly-ubiquitin accumulation and aggregation (as shown by IHC data) (Cannon et al., 2009).

Drolet and colleagues injected rats with rotenone (2.0 mg/kg, 1.0 ml/kg, i.p. 5 injections/week for 6 weeks) and found formic acid-resistant α-synuclein aggregates in the small intestine myenteric plexus, particularly 6-months after the last rotenone injection (3.5 median, vs 2.0 in Ctr) (Drolet et al., 2009). Mandel et al. injected male C57-BL mice with MPTP (24 mg/kg/day, ip for 5 days) and found α-synuclein aggregates (IHC data), which were decreased by using the radical scavengers apomorphine (injected s.c. at 10 mg/kg/day) or epigallocatechin-3-gallate (EGCG, given alone orally, 2 mg/kg/d) for 10 days) or a combination of both (Mandel et al., 2004).

Inhibition of the mitochondria respiratory chain induces oxidative stress that in turn leads to lipid peroxidation of cellular and vesicular membranes at synaptic sites, resulting in dysfunction of neurotransmitter release. These effects facilitate α-synuclein conformational changes, such as accumulation, and aggregation. It has been demonstrated that synaptic dysfunction (caused by mitochondrial dysfunction) triggered the accumulation of α-synuclein (Nakata et al., 2012). Also, alterations of mitochondrial fission or dynamics can reduce synaptic mitochondrial load and impair neuronal function by hindering the proper energy demand to ensure synaptic function. Mitochondrial behaviours, especially those regulated by neuronal activity and synapse location, determine their distribution in the axon (Obashi and Okabe, 2013). These observations support the idea that mitochondrial dysfunction can affect synaptic environment and consequently result in α-synuclein accumulation at synapses (Zaltieri et al., 2015).

• It was found that continuous administration of MPTP produced formation of nigral inclusions immunoreactive for ubiquitin and α-synuclein (Fornai et al., 2005). Mice were implanted with osmotic pump to deliver MPTP-HCl. Delayed and prolonged inhibition of striatal proteasome activity (i.e., 40-50-60% inhibition of UPS) occurred after continuous MPTP administration (respectively, 1-5-30 mg/kg MPTP daily) for the indicated time periods (Fig. 1) (Fornai et al, 2005). Continuous MPTP infusions caused also a long-lasting activation of glucose uptake. Additionally, in mice lacking α-synuclein, the MPTP-induced inhibition of the UPS system and the production of inclusion bodies were reduced (e.g., Ctr mice showed ~40% inhibition of postglutamyl peptidase (PGPH) activity, vs ~13% inhibition observed in α-synuclein KO mice) (Fig. 2), suggesting that α-synuclein could play an important role in UPS inhibition induced by MPP+ (Fornai et al., 2005). These data suggest that continuous, low-level exposure of mice to MPTP causes a Parkinson-like syndrome in a α-synuclein-dependent manner (Fornai et al., 2005).

These results are supported by other studies showing that α-synuclein−/− mice are resistant to MPTP toxicity (Dauer et al., 2002; Drolet et al., 2004). MPTP exposure (0.5, 5, 50 µM, 48 hr) increases in a dose-dependent manner the α-synuclein protein level in mesencephalic neurons in culture (e.g., ~70% increase at 5 µM vs Ctr) (Duka et al., 2006). Increased expression of α-synuclein predisposes DA neuronal cells to proteasomal dysfunction (~50% decrease compared to Ctr-vector cells) (Sun et al., 2005).

• Accumulation/overexpression of α-synuclein, both wild type and mutant, potentiates inhibition of proteasomal activity. Cells expressing mutant α-synuclein showed a reduction of lysosomal hydrolysis and chymotrypsin-like UPS function (by ~30%, compared to WT) (Stefanis et al., 2001).

• Proteasomal inhibition (by mean of lactacystin, a proteasome inhibitor, used at different concentrations for 24 hr) contributes to the accumulation of α-synuclein as it has been described by immunostaining in PC12 cells (Rideout et al., 2001) and in primary mesencephalic neurons (McNaught et al., 2002).

• α-Synuclein levels were selectively increased in the ventral midbrain (VMB) region of rotenone-infused rats with or without lesion (~ 110% increase vs Ctr) (Fig. 3) (Betarbet et al., 2006). Rotenone was administered up to 5 weeks, at 2.5 mg/kg/day. Additionally, 4 weeks of in vitro rotenone exposure (5 nM, on SK-N-MC human neuroblastoma cells) increased α-Synuclein levels by 24%, while lactacystin (9 μM, overnight) did not induce any detectable changes in α-synuclein levels. α-Tocopherol attenuated the rotenone-induced increase in α-synuclein (comparable to Ctr) (Fig. 4). Furthermore, levels of ubiquitinated proteins detected in solubilized protein fractions from SK-N-MC cells resulted increased (by 60%) with rotenone treatment (5 nM), and even more (by 484%) with rotenone combined with lactacystin (Fig. 5) (Betarbet et al., 2006).

• CI inhibition-induced proteasomal dysfunction has been reported in human SH-SY5Y neuroblastoma cells following acute rotenone exposure (Shamoto-Nagai et al., 2003). The proteasome activity decreased in the cells treated with rotenone (25 or 50 nM) in a time- and dose-dependent way. ATP addition restored the reduction of proteasome activity in the cells treated with 25 nM rotenone for 72 hr. However, after 96 hr of incubation with 25 or 50 nM rotenone, the activity was reduced respectively to 28.7% and 21.9% of control, and adding ATP did not increase the activity. After 120 hr, the activity was virtually undetectable (with or without added ATP) (Fig. 6). On the contrary, the levels of the proteins composing proteasome did not change with rotenone treatment (Shamoto-Nagai et al., 2003).

Cytoskeletal damage further enhances disturbed proteostasis:

• α-synuclein can trigger hyperphosphorylation of Tau. Treatment of primary mesencephalic neurons acutely (48 h) or subchronic treatment of wild-type (WT) mice with MPP+/MPTP results in selective dose-dependent hyperphosphorylation of Tau at Ser396/404 (p-Tau). The presence of α-synuclein was absolutely mandatory to observe MPP+/MPTP-induced increases in p-Tau levels, since no alterations in p-Tau were seen in transfected cells not expressing α-synuclein or in α-synuclein-/- mice. MPP⁺/MPTP also induced a significant accumulation of α-synuclein in both mesencephalic neurons and in WT mice striatum. Sub-chronic MPTP exposure increased phosphorylated-Tau in striatum of WT (but not α-Syn-/- mice) causing microtubule (MT) cytoskeleton instability that affects cellular microtubule transport (including axonal transport) (Qureshi et al., 2009; Duka et al., 2006). For instance, MPTP was found to elicit an increase of phosphorylated Tau at Ser262 by 2.8-, 4.5-, 4.6-, and 4.0-fold higher in 1, 5, 25, and 50 μM MPTP-treated cells than the basal level observed in Ctr/vehicle-treated cells, respectively. Additionally, MPTP caused a dose-dependent increase in the intracellular α-synuclein level in M17 human neuroblastoma cells (~3.5 fold increase in cells treated with 25 μM MPTP vs Ctr) (Qureshi and Paudel, 2009). These results were confirmed by other studies (e.g. Dauer et al., 2002; Drolet et al., 2004 etc.).

• α-synuclein accumulation followed by MT depolymerisation induces disruption in axonal transport, which leads to an accumulation of damaged organelles, aggregated/misfolded proteins and impaired vesicular release. Dopamine is leaking from the vesicles to the cytosol promoting an increase in oxidative stress, potentiated by dopamine oxidation (Feng, 2006; Kim et al., 2007). When microtubule network is disrupted, the amount of free tubulin increases, triggering α-synuclein fibrillization (Payton et al., 2001).

• Axonal transport might be impaired by misfolded α-synuclein through perturbation of microtubule assembly (Esposito et al., 2007; Lee et al., 2002; Chen et al., 2007 ), especially together with MAPT protein (Qureshi and Paudel, 2011; Giasson et al., 2003). It induces not only microtubule disruption but also impairs microtubule-dependent trafficking (Lee at al., 2006). MT-dependent transport is important for maintaining the Golgi structure, and thus, depolymerization of the MT leads to a specific pattern of Golgi fragmentation (Cole et al., 1996). When the MT network was disrupted by nocodazole treatment (5 µg/mL) or α-synuclein was overexpressed, this normally compact organelle was fragmented and dispersed (IC images) as shown in COS-7 cells (Lee at al., 2006). Similarly, overexpression of α-synuclein in differentiated SH-SY5Y cells caused Golgi fragmentation (e.g., ~190% increased fragmented Golgi at 12 m.o.i. (multiplicity of infection) of α-synuclein vs Ctr) (Lee at al., 2006).

• It was found that α-synuclein mutants associated with PD exhibit reduced transport in neurons, as shown in rat primary neuronal cortical cultures transfected with wild-type (WT), A53T or A30P α-synuclein. For instance, the rate of transport (expressed in µm/hr) was reduced of ~55% and ~60% after 3-4 hr for A30P and A53T respectively (vs Ctr-WT) (Saha et al., 2004).

• Damaged cytoskeletal proteins disrupt also mitochondrial trafficking. Mitochondria use cytoskeletal proteins as tracks for their directional movement (Nogales, 2000). The cytoskeletal system regulates not only mitochondrial movement but also their morphology and function. Therefore, damage to microtubules perturbs transport of mitochondria through axons, increasing their retrograde movement. These changes in mitochondria dynamics lead to a decrease of mitochondria numbers in axons and mitochondria accumulation in cell bodies (De vos et al., 2007; Miller and Sheetz, 2004). Depletion of mitochondria quantity and function in axons occurs in neurodegenerative disorders (Brownlees et al., 2002; Stamer et al., 2002). Since mitochondria are ATP suppliers and microtubules need ATP to accomplish their function, mitochondrial dysfunction has a profound effect on axonal transport and function (De Vos et al., 2008).

• Mitochondrial dysfunction may damage mitochondrial trafficking through calcium dysregulation. Cytosolic Ca2+ is one of the best-studied regulators of mitochondrial movement. Elevation of cytosolic Ca2+ stops both the anterograde and retrograde trafficking of mitochondria in neurons and in many cell lines. (Chang et al. 2006; Szabadkai et al. 2006). In H9c2 cells simultaneous measurements of free Ca2+ levels and mitochondrial dynamics showed that 50% reductions in mitochondrial movement occurred at concentrations of approximately 400 nM Ca2+, and a complete arrest in the low micromolar range (Yi et al. 2004; Saotome et al., 2008). These are indirect proofs suggesting that inhibition of CI, followed by mitochondrial dysfunction, could damage mitochondrial trafficking. Also, chronic exposure to rotenone (50 nM at different times of exposure) was reported to reduce mitochondrial movement in differentiated SH-SY5Y cells (e.g., ~30% reduction of mitochondrial movement (µm/sec) after 8 days of rotenone treatment vs Ctr) (Borland et al., 2008).

Human studies

• In PD patient postmortem cortical tissues, levels of oligomeric α-synuclein in SNpc (~1000% vs Ctr samples) and expression of LC3-II levels (~130% vs Ctr samples) were up-regulated (Yu et al., 2009) (for further info, see the review from Vekrellis et al., 2011).

• The pathological observations in PD autopsy brains showed that LC3-II levels were elevated in the SNpc and amygdala of PD brain samples, suggesting an increase in macroautophagy (but they did not reach statistical significance). LC3 colocalized with α-synuclein in most LBs and Lewy neurites in PD SNpc as well as in small punctate α-synuclein immunoreactive inclusions (IC images) (Alvarez-Erviti et al., 2010).

• Analogously, another study reported that brain homogenates derived from the temporal cortex of dementia with LB (DLB) patients vs non-demented controls were characterized by higher levels of both mTor (~130% vs Ctr) and p-mTor (~ 10 folds higher than Ctr), and levels of Atg7 (molecular initiator of autophagy) were moderately reduced in DLB cases compared to Ctr (~ 40% lower than Ctr). Consistent with the studies in human brains, levels of both mTor and p-mTor were increased in the membrane fractions from brains of α-synuclein tg mice compared to non tg controls (respectively, by ~250% and ~200% vs Ctr), and levels of Atg7 were reduced in α-synuclein tg brains compared to non tg controls (~75% less than Ctr) (Crews et al., 2010).

• Another study showed that post-mortem brain samples derived from PD patients, compared to age-matched controls, presented significant reductions of LAMP1 (2069.10 ± 329.52), CatD (1809.35 ± 533.47), HSP73 (2604.92 ± 494.56), and 20S proteasome (1660.84 ± 229.87) calculated by optic density (OD) measures (Chu et al., 2009). These data globally indicate that the functions of both the UPS and ALP systems is compromised in PD patients.

• The exact molecular link from mitochondrial dysfunction to disturbed proteostasis is not known. It is not clear which is the oxidative modification that drives the process.

• The sequence of events taking place after inhibition of CI is not entirely clear (Zaltieri et al., 2015). Some studies suggest that induced oxidative stress leads to α-synuclein aggregation that triggers proteosomal dysfunction (Betarbet et al., 2006). Such order of events is suggested to take place in vivo (McNaught and Jenner, 2001). However, in other studies opposite sequence of events is proposed suggesting that first proteosomal dysfunction take place that leads to α-synuclein aggregation.

A vicious circle is observed here as α-synuclein aggregation potentiates proteosomal dysfunction and v/v. In this vicious cycle it is difficult to establish exact quantitative relationship of these two events.

• Whether α-synuclein is a substrate for proteasome remains controversial since both positive and negative data have been reported (Paxinou et al., 2001). Furthermore, polyubiquitination of α-synuclein, a prerequisite for 26S proteasomal degradation has yet to be reported (Stefanis et al., 2001). It is also not clear whether polyubiquitination of α-synuclein is necessary for its degradation. However, α-synuclein gets targeted by the UPS in the SHSY5Y neuroblastoma cell line. Phosphorylated α-synuclein gets targeted to mono- or di-ubiquitination in synucleinopathy brains (Hasegawa et al., 2002), but it is not clear if this modification can play any role in proteasomal degradation since monoubiquitination of proteins serves mainly as a signal for endocytosis or membrane trafficking.

• On the contrary to the increased α-synuclein levels observed in the midbrain, decreased α-synuclein levels were found in the cerebellums of PD patients when compared to controls, suggesting an imbalance of α-synuclein levels in different parts of the brain (Westerlund et al., 2008).

• Although mitochondrial alterations have been reported in PD patients (Ikawa et al., 2011) and disease models, it is not clear whether they represent a primary pathogenic mechanism. In particular, the critical interplay between mitochondrial dysfunction and oxidative stress, which has been widely reported in PD (Dias et al., 2013) and could constitute either a cause or a consequence of mitochondrial damage, hampers an effective comprehension of the above mentioned studies. Oxidative stress can constitute a bridge connecting mitochondrial dysfunction to the induction of α-synuclein misfolding, aggregation, and accumulation, but otherwise it may be also triggered by these latter events that in turn could induce mitochondrial alterations (Zhu and Chu, 2010; Dias et al., 2013).

• It is still unclear whether the involvement of α-synuclein in chronic MPTP toxicity reflects a physiological function for α-synuclein that has been activated in the wrong context, or whether α-synuclein produces an accidental pathogenicity that contributes to MPTP toxicity but is unrelated to the normal function of α-synuclein (Fornai et al., 2005).

• The inconsistent effects of MPP+ on autophagy (up or down regulation) are reported. It may be attributed to differences observed between immortalized cell lines and primary neurons, different timing or dose. While dysregulation of autophagy is always described, the direction is not clear. Further studies are required to clarify this issue.

• MPTP administration does not induce Lewy body formation (in contrast to rotenone) characteristic of PD, even after repeated injections (Drolet et al., 2004; Dauer et al., 2002).

• There is also controversy over whether the increase in autophagic markers is protective or, on the contrary, causative of neuronal death.

• MPP+ may have effects apart from CI inhibition, e.g., on microtubules but it is still unclear whether this is a primary effect. Indeed, MPP+ binds to microtubules in PC12 cells and inhibits their polymerization and stability (Cappelletti et al., 1999; Cappelletti et al., 2001).

• It is not clear whether microtubules disruption may be associated with α-synuclein aggregation since tubulin was shown to co-localize with α-synuclein in Lewy bodies. Furthermore, tubulin folding is dependent on ATP and GTP hydrolysis, and mitochondrial dysfunction with subsequent energy failure could trigger microtubules disruption. Cytoskeletal microtubule (MT) injury is likely to be responsible for altered rearrangement and movement of cell organelles, being a common feature of several neurodegenerative diseases including PD (Wade, 2009; Mattson et al., 1999).

• It is not clear whether rotenone could cause microtubules depolymerization in vivo and in vitro (Brinkley et al., 1974) by binding to the colchicine site on tubulin heterodimers (Marshall et al., 1978). Ren and Feng (2007) found that microtubule depolymerization induced by rotenone caused vesicle accumulation in the soma and kills neurons.