The NRF2-p97-NRF2 negative feedback loop.

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.

The dark side of NRF2 in arsenic carcinogenesis.

Arsenic is an environmental toxicant that significantly enhances the risk of developing disease, including several cancers. While the epidemiological evidence supporting increased cancer risk due to chronic arsenic exposure is strong, therapies tailored to treat exposed populations are lacking. This can be accredited in large part to the chronic nature and pleiotropic pathological effects associated with prolonged arsenic exposure. Despite this fact, several putative mediators of arsenic promotion of cancer have been identified. Among these, the critical transcription factor NRF2 has been shown to be a key mediator of arsenic's pro-carcinogenic effects. Importantly, the dependence of arsenic-transformed cancer cells on NRF2 upregulation exposes a targetable liability that could be utilized to treat arsenic-promoted cancers. In this chapter, we briefly introduce the "light" vs "dark" side of the NRF2 pathway. We then give a brief overview of arsenic metabolism, and discuss the epidemiological and experimental evidence that support arsenic promotion of different cancers, with a specific emphasis on mechanisms mediated by chronic, non-canonical activation of NRF2 (i.e., the "dark" side). Finally, we briefly highlight how the non-canonical NRF2 pathway plays a role in other arsenic-promoted diseases, as well as research directions that warrant further investigation.

NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8.

Enhancing the intracellular labile iron pool (LIP) represents a powerful, yet untapped strategy for driving ferroptotic death of cancer cells. Here, we show that NRF2 maintains iron homeostasis by controlling HERC2 (E3 ubiquitin ligase for NCOA4 and FBXL5) and VAMP8 (mediates autophagosome-lysosome fusion). NFE2L2/NRF2 knockout cells have low HERC2 expression, leading to a simultaneous increase in ferritin and NCOA4 and recruitment of apoferritin into the autophagosome. NFE2L2/NRF2 knockout cells also have low VAMP8 expression, which leads to ferritinophagy blockage. Therefore, deletion of NFE2L2/NRF2 results in apoferritin accumulation in the autophagosome, an elevated LIP, and enhanced sensitivity to ferroptosis. Concordantly, NRF2 levels correlate with HERC2 and VAMP8 in human ovarian cancer tissues, as well as ferroptosis resistance in a panel of ovarian cancer cell lines. Last, the feasibility of inhibiting NRF2 to increase the LIP and kill cancer cells via ferroptosis was demonstrated in preclinical models, signifying the impact of NRF2 inhibition in cancer treatment.

Decreased autophagosome biogenesis, reduced NRF2, and enhanced ferroptotic cell death are underlying molecular mechanisms of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Caloric excess and sedentary lifestyles have led to an epidemic of obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD). The objective of this study was to investigate the mechanisms underlying high fat diet (HFD)-induced NAFLD, and to explore NRF2 activation as a strategy to alleviate NAFLD. APPROACH AND RESULTS: Herein, we demonstrated that high fat diet (HFD) induced lipid peroxidation and ferroptosis, both of which could be alleviated by NRF2 upregulation. Mechanistically, HFD suppressed autophagosome biogenesis through AMPK- and AKT-mediated mTOR activation and decreased ATG7, resulting in KEAP1 stabilization and decreased NRF2 levels in mouse liver. Furthermore, ATG7 is required for HFD-induced NRF2 downregulation, as ATG7 deletion in Cre-inducible ATG7 knockout mice decreased NRF2 levels and enhanced ferroptosis, which was not further exacerbated by HFD. This finding was recapitulated in mouse hepatocytes, which showed a similar phenotype upon treatment with saturated fatty acids (SFAs) but not monounsaturated fatty acids (MUFAs). Finally, NRF2 activation blocked fatty acid (FA)-mediated NRF2 downregulation, lipid peroxidation, and ferroptosis. Importantly, the HFD-induced alterations were also observed in human fatty liver tissue samples. CONCLUSIONS: HFD-mediated autophagy inhibition, NRF2 suppression, and ferroptosis promotion are important molecular mechanisms of obesity-driven metabolic diseases. NRF2 activation counteracts HFD-mediated NRF2 suppression and ferroptotic cell death. In addition, SFA vs. MUFA regulation of NRF2 may underlie their harmful vs. beneficial effects. Our study reveals NRF2 as a key player in the development and progression of fatty liver disease and that NRF2 activation could serve as a potential therapeutic strategy.

NRF2 and Diabetes: The Good, the Bad, and the Complex.

Despite decades of scientific effort, diabetes continues to represent an incredibly complex and difficult disease to treat. This is due in large part to the multifactorial nature of disease onset and progression and the multiple organ systems affected. An increasing body of scientific evidence indicates that a key mediator of diabetes progression is NRF2, a critical transcription factor that regulates redox, protein, and metabolic homeostasis. Importantly, while experimental studies have confirmed the critical nature of proper NRF2 function in preventing the onset of diabetic outcomes, we have only just begun to scratch the surface of understanding the mechanisms by which NRF2 modulates diabetes progression, particularly across different causative contexts. One reason for this is the contradictory nature of the current literature, which can often be accredited to model discrepancies, as well as whether NRF2 is activated in an acute or chronic manner. Furthermore, despite therapeutic promise, there are no current NRF2 activators in clinical trials for the treatment of patients with diabetes. In this review, we briefly introduce the transcriptional programs regulated by NRF2 as well as how NRF2 itself is regulated. We also review the current literature regarding NRF2 modulation of diabetic phenotypes across the different diabetes subtypes, including a brief discussion of contradictory results, as well as what is needed to progress the NRF2 diabetes field forward.

CHML is an NRF2 target gene that regulates mTOR function.

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is often highly expressed in non-small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM-like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (-1622 to -1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA-mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2-Rep2-dependent regulation of mTOR function.

NRF2 Loss Accentuates Parkinsonian Pathology and Behavioral Dysfunction in Human α-Synuclein Overexpressing Mice.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a central regulator of cellular stress responses and its transcriptional activation promotes multiple cellular defense and survival mechanisms. The loss of NRF2 has been shown to increase oxidative and proteotoxic stress, two key pathological features of neurodegenerative disorders such as Parkinson's disease (PD). Moreover, compromised redox homeostasis and protein quality control can cause the accumulation of pathogenic proteins, including alpha-synuclein (α-Syn) which plays a key role in PD. However, despite this link, the precise mechanisms by which NRF2 may regulate PD pathology is not clear. In this study, we generated a humanized mouse model to study the importance of NRF2 in the context of α-Syn-driven neuropathology in PD. Specifically, we developed NRF2 knockout and wild-type mice that overexpress human α-Syn (hα-Syn+/Nrf2-/- and hα-Syn+/Nrf2+/+ respectively) and tested changes in their behavior through nest building, challenging beam, and open field tests at three months of age. Cellular and molecular alterations in α-Syn, including phosphorylation and subsequent oligomerization, as well as changes in oxidative stress, inflammation, and autophagy were also assessed across multiple brain regions. It was observed that although monomeric α-Syn levels did not change, compared to their wild-type counterparts, hα-Syn+/Nrf2-/- mice exhibited increased phosphorylation and oligomerization of α-Syn. This was associated with a loss of tyrosine hydroxylase expressing dopaminergic neurons in the substantia nigra, and more pronounced behavioral deficits reminiscent of early-stage PD, in the hα-Syn+/Nrf2-/- mice. Furthermore, hα-Syn+/Nrf2-/- mice showed significantly amplified oxidative stress, greater expression of inflammatory markers, and signs of increased autophagic burden, especially in the midbrain, striatum and cortical brain regions. These results support an important role for NRF2, early in PD progression. More broadly, it indicates NRF2 biology as fundamental to PD pathogenesis and suggests that targeting NRF2 activation may delay the onset and progression of PD.

MGST1, a new soldier of NRF2 in the battle against ferroptotic death.

In this issue of Cell Chemical Biology, Kuang et al. (2021) identify microsomal glutathione-S-transferase 1 (MGST1) as an NRF2 target gene that suppresses ferroptosis in pancreatic cancer cells. Mechanistically, MGST1 binds ALOX5 during ferroptosis induction, inhibiting lipid peroxide production. Thus, MGST1 could represent a viable therapeutic target for treating pancreatic cancer.

Enhanced NRF2 expression mitigates the decline in neural stem cell function during aging.

Although it is known that aging affects neural stem progenitor cell (NSPC) biology in fundamental ways, the underlying dynamics of this process are not fully understood. Our previous work identified a specific critical period (CP) of decline in NSPC activity and function during middle age (13-15 months), and revealed the reduced expression of the redox-sensitive transcription factor, NRF2, as a key mediator of this process. Here, we investigated whether augmenting NRF2 expression could potentially mitigate the NSPC decline across the identified CP. NRF2 expression in subventricular zone (SVZ) NSPCs was upregulated via GFP tagged recombinant adeno-associated viral vectors (AAV-NRF2-eGFP), and its cellular and behavioral effects compared to animals that received control vectors (AAV-eGFP). The vectors were administered into the SVZs of aging rats, at time points either before or after the CP. Results indicate that animals that had received AAV-NRF2-eGFP, prior to the CP (11 months of age), exhibited substantially improved behavioral function (fine olfactory discrimination and motor tasks) in comparison to those receiving control viruses. Further analysis revealed that NSPC proliferation, self-renewal, neurogenesis, and migration to the olfactory bulb had significantly increased upon NRF2 upregulation. On the other hand, increasing NRF2 after the CP (at 20 months of age) produced no notable changes in NSPC activity at either cellular or behavioral levels. These results, for the first time, indicate NRF2 pathway modulation as a means to support NSPC function with age and highlight a critical time-dependency for activating NRF2 to enhance NSPC function.

An NRF2 Perspective on Stem Cells and Ageing.

Redox and metabolic mechanisms lie at the heart of stem cell survival and regenerative activity. NRF2 is a major transcriptional controller of cellular redox and metabolic homeostasis, which has also been implicated in ageing and lifespan regulation. However, NRF2's role in stem cells and their functioning with age is only just emerging. Here, focusing mainly on neural stem cells, which are core to adult brain plasticity and function, we review recent findings that identify NRF2 as a fundamental player in stem cell biology and ageing. We also discuss NRF2-based molecular programs that may govern stem cell state and function with age, and implications of this for age-related pathologies.

Breakdown of an Ironclad Defense System: The Critical Role of NRF2 in Mediating Ferroptosis.

Ferroptosis is a non-apoptotic mode of regulated cell death that is iron and lipid peroxidation dependent. As new mechanistic insight into ferroptotic effectors and how they are regulated in different disease contexts is uncovered, our understanding of the physiological and pathological relevance of this mode of cell death continues to grow. Along these lines, a host of pharmacological modulators of this pathway have been identified, targeting proteins involved in iron homeostasis; the generation and reduction of lipid peroxides; or cystine import and glutathione metabolism. Also, of note, many components of the ferroptosis cascade are target genes of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), indicating its critical role in mediating the ferroptotic response. In this review, we discuss the in vitro, in vivo, and clinical evidence of ferroptosis in disease, including a brief discussion of targeting upstream mediators of this cascade, including NRF2, to treat ferroptosis-driven diseases.

Metabolic Investigations of the Molecular Mechanisms Associated with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies) and the associated loss of dopaminergic cells in the substantia nigra. Mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences. Exposure to environmental toxicants including pesticides and metals (e.g., paraquat (PQ) and manganese (Mn)) is also recognized as an important PD risk factor. Thus, aging, genetic alterations, and environmental factors all contribute to the etiology of PD. In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear. In this study, we summarize our findings to date regarding the toxic synergistic effect between α-synuclein and paraquat treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat treatment that is enhanced by the overexpression of α-synuclein. PQ "hijacks" the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMP-activated protein kinase (AMPK) activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism, likely directing additional carbon to the PPP to supply paraquat redox cycling.

Metabolic Dysfunction in Parkinson's Disease: Bioenergetics, Redox Homeostasis and Central Carbon Metabolism.

The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the accumulation of protein inclusions (Lewy bodies) are the pathological hallmarks of Parkinson's disease (PD). PD is triggered by genetic alterations, environmental/occupational exposures and aging. However, the exact molecular mechanisms linking these PD risk factors to neuronal dysfunction are still unclear. Alterations in redox homeostasis and bioenergetics (energy failure) are thought to be central components of neurodegeneration that contribute to the impairment of important homeostatic processes in dopaminergic cells such as protein quality control mechanisms, neurotransmitter release/metabolism, axonal transport of vesicles and cell survival. Importantly, both bioenergetics and redox homeostasis are coupled to neuro-glial central carbon metabolism. We and others have recently established a link between the alterations in central carbon metabolism induced by PD risk factors, redox homeostasis and bioenergetics and their contribution to the survival/death of dopaminergic cells. In this review, we focus on the link between metabolic dysfunction, energy failure and redox imbalance in PD, making an emphasis in the contribution of central carbon (glucose) metabolism. The evidence summarized here strongly supports the consideration of PD as a disorder of cell metabolism.

Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions.

While environmental exposures are not the single cause of Parkinson's disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by the paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and in the levels of glucose transporter type 4 (GLUT4) and Na+-glucose transporters isoform 1 (SGLT1) proteins, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose accumulation and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism/transport and the pentose phosphate pathway (6-aminonicotinamide). These results demonstrate that glucose metabolism and AMPK regulate dopaminergic cell death induced by gene (α-synuclein)-environment (PQ) interactions.

Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways.

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson's disease (PD). Ubiquitin (Ub), alpha (α)-synuclein, p62/sequestosome 1, and oxidized proteins are the major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effects of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP(+), or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ-induced cell death. The inhibition of proteasomal activity by PQ was found to be a late event in cell death progression and had neither effect on the toxicity of either MPP(+) or PQ, nor on the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins), and carbonylated proteins induced by PQ. PQ- and MPP(+)-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagy. We confirmed that PQ and MPP(+) impaired autophagy flux and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane-associated foci in yeast cells. Our results demonstrate that the inhibition of protein ubiquitination by PQ and MPP(+) is involved in the dysfunction of Ub-dependent protein degradation pathways.

Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability.

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism - intramitochondrial quality control (IMQC) - is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.