Alpha-synuclein nitration and its implications in Parkinson's disease

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Alpha-synuclein nitration and its implications in Parkinson’s disease Yi xi He, Zhong wang Yu, and Sheng-Di Chen ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00288 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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ACS Chemical Neuroscience

Alpha-synuclein nitration and its implications in Parkinson’s disease Yixi He1, Zhongwang Yu2,*, Shengdi Chen1,* 1

Department of Neurology and Institute of Neurology, Ruijin Hospital affiliated

to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. 2

Institute of Neuroscience, Second Military Medical University, Shanghai 200433, China.

*Correspondence to Shengdi Chen, E-mail:[email protected], or Zhongwang Yu, E-mail:[email protected].

Abstract Parkinson’s disease is pathologically characterized by the degeneration of dopaminergic neurons in the substantia nigra and the accumulation of neuronal cytoplasmic inclusions known as Lewy bodies, which are primarily composed of α-synuclein. Post-translational modifications of α-synuclein induced by nitrative stress have been linked to neurodegeneration. Here, we review the concept of α-synuclein nitration and its biological consequences. We also discuss the pathological roles of nitrated α-synuclein and their potential clinical implications in Parkinson’s disease.

Key words: Parkinson disease; α-synuclein; nitration

Introduction Parkinson's disease (PD) is one of the common neurodegenerative diseases, with a prevalence of 2-3% of the population over 65 years old (1). The neuropathological hallmarks of PD include striatal dopamine deficiency caused by the degeneration of dopaminergic neurons in the substantia nigra and the presence of neuronal cytoplasmic inclusions, termed Lewy bodies, that are mainly composed of deposited α-synuclein (ASN) (1). Multiple mechanisms underlying ASN deposition have been presented, such as overexpression, misfolding, post-translational processing abnormalities, etc (1-3). ASN can ACS Paragon Plus Environment

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undergo a variety of post-translational modifications, such as phosphorylation, ubiquitination, glycosylation, etc. Among them, nitration is a type of modification induced by nitrative stress. Here, we provide a progress update on our understanding of the biological effects and prevailing hypotheses regarding the nitration of ASN in the pathogenesis of PD, aiming to provide novel clues to explicate the pathogenesis, diagnosis and treatment of Parkinson's disease. 1. ASN nitration and its biological consequences 1.1 ASN structure and nitration sites The synuclein family has three subtypes, α, β and γ, and ASN is the main subtype. Though the physiological function of ASN has not been fully elucidated, it has been extensively documented to play a crucial role in synaptic vesicle trafficking and mitochondrial function regulation and serves as a potential molecular chaperone (2, 4). Human ASN consists of 140 amino acid residues and can be divided into the N-terminal (1-60AA), central (61-95 AA) and C-terminal (96-140 AA) portions (5). The N-terminus can adopt an amphipathic helical structure that may serve as/mimic a mitochondrial targeting sequence peptide, potentially related to mitochondrial dysfunction; the central, hydrophobic region plays a crucial role in ASN fibrillary aggregation and the formation of Lewy bodies. The C-terminal is rich in acidic amino acid residues. This C-terminal region has been proposed to be crucial for the solubility and stability of ASN and serves as a domain for interactions with many other proteins. ASN undergoes a variety of post-translational modifications, such as phosphorylation, ubiquitination, and nitration. Among them, nitration is an undesirable modification as nitrated ASN is the biomarker associated with oxidative and nitrative damage. A wide range of ASN nitration modifications can be detected in Lewy bodies in patients with neurodegenerative diseases, such as PD, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, and multiple system atrophy (6). In this form of post-translational modification, the nitro group (-NO2) is added to replace a hydrogen atom in the 3’ position of the tyrosine phenolic ring to form 3-nitrotyrosine (7). ASN has four tyrosine

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residues, residues Y39, Y125, Y133, and Y136, located in the N-terminal region and the C terminal region, that are all susceptible to nitration (5, 7). 1.2 Effect of nitration on ASN aggregation The misfolding and aggregation of ASN is an important mechanism of the progressive degeneration of dopaminergic neurons. Under normal physiological conditions, ASN forms helical folded tetramers and is able to counteract aggregation (8). Although the reason for the misfolding of ASN is not yet fully identified, post-translational modifications of ASN, particularly nitrative modifications, may have an important role in its misfolding (5, 7, 9). Treatment of recombinant human ASN with a nitration reagent can induce the formation of ASN oligomers, through the formation of covalently crosslinked dityrosine, which is not soluble in sodium dodecyl sulfonate solution and has high thermal stability (10). This study of ASN nitration revealed a great deal of information regarding its impact on aggregation and the formation of LBs. Using HEK 293 cells stably transfected with wild-type and mutant ASN, Paxinou E et al. found that the intracellular generation of nitrating agents results in the formation of ASN aggregates, although the aggregates formed in cells expressing the A53T mutant were higher than in those expressing the A30P mutant (11). As PD-related neuroinflammation is accompanied by high expression of inducible nitric oxide synthase and elevated NO levels, Stone DK et al. demonstrated that overexpression of nitric oxide synthase promoted nitration of ASN and oligomerization of ASN in neurons (12). On the other hand, overexpression of monoamine oxidase B resulted in intracellular overloaded of ASN and a 9-fold increase in the nitrated level of ASN 39Y, whereas the nitrated level at sites Y125, Y133 and Y136 did not change significantly; accordingly, deprenyl, an inhibitor of MAO-B, eliminated this effect (13). These studies further revealed the essential role and distinct features of cellular nitrative and oxidative stress in ASN nitration and aggregation. The nitrative modifications of ASN may have a great impact on ASN structure and oligomerization. For example, ASNs can dimerize through nitrated tyrosine to form dimers, and Y125 plays a critical role in nitrated stress-induced ASN dimerization (14). Hodara R and colleagues used chromatographically purified nitrated ASN monomers, dimers and oligomers to determine the ef-



fects of tyrosine nitration on ASN fibrillation (15). These researchers found that nitrated ASN monomers and dimers at low concentrations promoted the formation of ASN fibrils rather than oligomers. By using immunoelectron microscopy analysis, they revealed that nitrated ASN monomers and dimers were able to incorporate into fibrils. Interestingly, the purified nitrated ASN monomer did not form fibrils by itself. Uversky VN et al., using biochemical and biophysical techniques, examined the effect of nitration on the fibril formation of recombinant human ASN. They found that nitration caused a partially folded, intrinsically disordered conformation of ASN monomers. Although the nitrated ASN presented as monomers under acidic conditions, it tended to form oligomers under pH-neutral conditions (16). By using native chemical ligation combined with a novel desulfurization strategy, Burai R et al. explored the site-specific incorporation of 3-nitrotyrosine at different regions of ASN to investigate the role of nitration at single or multiple tyrosine residues in regulating ASN structure, membrane binding, oligomerization, and fibril formation (17). These authors demonstrated that the intermolecular interactions between the N- and C- terminal regions play crucial roles in mediating nitrationinduced ASN oligomerization. ASN is a substrate of the protein-degrading enzyme, calpain I, and calpain cleavage sites are in the middle region of soluble ASN. Degradation experiments have shown that nitrated ASN monomers have a lower rate of degradation than control; however, the fragments of this degradation were unable to self-fibrillize but prevented full-length ASN from fibrillization (18). However, calpain-mediated cleavage of fibrils composed of ASN or nitrated ASN generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length ASN to co-assemble. The phosphorylation of serine 129 of ASN has been shown to modulate the autophagic clearance of inclusions. Kleinknecht A et al. found that the C-terminal Y133 of ASN was required for the protective phosphorylation of S129, whereas Y133 nitration of ASN impeded the clearance of ASN aggregates (19). These studies provide a new perspective underlying the mechanism by which nitration contributes to ASN aggregation. 1.3 Effect of ASN nitration on membrane-binding capacity


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Membrane binding is mediated by the 1-95 region of ASN, while nitration of Y39 within this region interferes with the binding of ASN to membrane lipids through the charge-repulsion effect. Sevcsik E et al. analysed the effect of nitration of ASN on the membrane-binding ability and found that nitration of Y125, Y133 and Y136 at the C-terminal region also effectively inhibited binding; further studies indicated that nitration of the C-terminal region altered the conformation of ASN, thereby interfering in its binding capacity with membrane lipids (20). The ability of nitrated ASN to display an alpha helical conformation was disrupted in the presence of liposomes, which was associated with a decrease in the affinity of ASN monomers to vesicles as a result of the nitration of Y39 (15). Trostchansky A et al. demonstrated that the association of ASN to membrane lipids protected the protein from oxidation and nitration and thus diminished the formation of protein molecules capable of forming aggregates; however, ASN could be modified by products of lipid peroxidation to form new adducts (21). 2.The pathological roles of nitrated ASN in PD 2.1 The nitration of ASN in the Parkinsonian brain Many neurodegenerative disorders, especially those with synucleinopathies, are characterized by ASN aggregation. By immunostaining with nitrotyrosine antibodies, Giasson BI et al. found that nitrated ASN existed extensively in the brains of patients with neurodegenerative diseases such as PD, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, and multiple system atrophy (6). This nitrated ASN was present not only in the neurofibrillary tangles of the inclusion body but also in the insoluble components of brain regions involved in synucleinopathies. Duda JE et al. also demonstrated wide spread nitration of ASN in pathological inclusions of various neurodegenerative synucleinopathies (22). ASN nitration in the brain provides direct evidence for the association of oxidative and nitrative stress with the occurrence and development of neurodegenerative diseases. To clarify whether there were any differences on ASN expression and posttranslational modifications in PD animal models, researchers examined the MPTP-induced PD mouse model. ASN but not β-synuclein or synaptophysin



was found to be nitrated in the striatum and ventral midbrain (23). ASN expression was greatly up-regulated after MPTP was injected into the substantia nigra of squirrel monkeys, while β-synuclein and synaptophysin were unchanged (24). Moreover, ASN deposition with nitrative modification was shown in the soma of midbrain neurons and axons. These intriguing results indicate that ASN-specific nitration correlates with MPTP toxicity and DArelated neuronal degeneration. Neuroinflammation is one of the major mechanisms of PD-related DA neuronal lesions (25, 26). Gao HM et al. studied the relationship between ASN dysfunction and neuroinflammation by stereotaxic injection of lipopolysaccharide (LPS), a proinflammatory agent, into the substantia nigra of ASN transgenic mice (27). They found that ASN within intracellular inclusions was nitrated, and neuroinflammation was closely associated with ASN deposition and DA neuron death. They further demonstrated that the reduction in NO and peroxide produced by microglia contributed to the neuroprotection observed. Using transcriptomic and proteomic technology, Reynolds found that nitrated ASN potently activated microglia and produced a large number of inflammatory mediators, leading to dopaminergic neuron death (28). Further studies from the same laboratory revealed that immunization of mice with nitrated ASN activated Th17, causing microglial activation and the release of large amounts of reactive oxygen species and reactive nitrogen, potentially leading to more ASN nitration (29, 30). Ageing is one unequivocal risk factor in the pathogenesis of PD (31), suggesting that ageing may have an effect on the nitration of ASN. McCormack AL et al. reported that dopaminergic cell bodies immunoreactive for nitrated ASN were rarely observed in adult mature animals but were significantly more frequent in the substantia nigra of old monkeys (32). Dopaminergic neurons in the substantia nigra are peculiarly vulnerable to metabolic and oxidative stress，as they exhibit autonomous pacemaking activity and possess long axons which require great energy to supply, and high levels of cytosolic dopamine with its metaboites can cause toxic oxidative stress (1, 33, 34). Therefore, the increase might be associated with an increased amount of unmodified ASN and the prooxidant environment that characterizes older neurons in


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the substantia nigra (32, 35). Choi DY et al. demonstrated that stereotaxic injection of LPS in the inner globus pallidus activated microglia, significantly facilitating proinflammatory cytokine expression, especially in older rats; compared with young rats, aged animals expressed significantly elevated expression levels of both NO synthase and ASN nitration (36). These results suggest that ageing may boost neuroinflammation, enhance the nitration of ASN, and cause DA neuron degeneration. 2.2 Impact of nitrated ASN on DA neurons Although much evidence has hinted that ASN nitration correlates with neuronal degeneration, the precise cause-effect relationship between the nitration of ASN and the survival of DA neurons was unclear for a long time. Shavali S et al. examined the effects of activated macrophage-derived nitric oxidedependent oxidative stress on neuronal degeneration (37). They found that when proinflammatory macrophages produced nitric oxide and peroxynitrite, accompanied by increased levels of peroxynitrite and elevated nitrated ASN, SH-SY5Y cell death increased, whereas the use of iNOS inhibitors ameliorated ASN nitration and hampered SH-SY5Y cell death. This study suggests that activated microglia can induce oxidative stress in dopaminergic neurons, resulting in the nitration of ASN and subsequently DA neuronal death. Liu Y et al. found that nitrated ASN with four 3-nitrotyrosines (Tyr39, Tyr125, Tyr133, and Tyr136) existed as a mixture of monomers, dimers, and polymers in solution, and the dimers and polymers of the nitrated ASN induced dosedependent SH-SY5Y cell cytotoxicity (38). Interestingly, treatment with the anti-integrin α5β1 antibody ameliorated the cellular damage caused by nitrated ASN; further biochemical experiments illustrated that nitrated ASN could bind to the integrin on the cell membrane, thus inducing cytotoxicity presumably via the integrin-iNOS/-FAK signalling pathway (38). We previously constructed, purified and transduced nitrated ASN into dopaminergic cells, and scrutinized the effects of nitrated ASNs on DA neurons in vitro and in vivo (39). The results illustrated that nitrated ASN was cytotoxic to SH-SY5Y cells and primary midbrain DA neurons; administration of nitrated ASN in the rat substantia nigra caused severe DA neuron loss and subsequently led to up-regulation of D2R in the striatum. Compared to ASN injec-



tion, stereotaxic injection of nitrated ASN to the rat substantia nigra induced more PD-like behaviour changes in animals, such as bradykinesia and postural instability. These observations indicated that nitrated ASN induces DA neuron degeneration and may be a major factor in the pathogenesis of PD. PD is pathologically characterized by the degeneration of dopaminergic neurons in the substantia nigra and the accumulation of ASN within neurons. It has been hypothesized that neuronal damage in PD occurs in an ASN spreading and ascending pattern from peripheral nerves to the brainstem and midbrain (40). In contrast to this ascending theory, Engelender S and Isacson O recently proposed the threshold theory (41), suggesting that a simultaneous pathology developing in multiple systems and systems reach their individual thresholds for symptoms at different rates, providing a more accurate explanation for the onset and progression of PD. According to this view, disease mechanisms simultaneously influence different neurons, and the death and dysfunction of neurons in a particular area depend on their individual vulnerability (41). In DA neurons of the substantia, it is likely that the prooxidant environment would promote the nitration of ASN, resulting in the formation of aggregates and causing the cytotoxicity. Meanwhile, the activation of neuroinflammation would boost the release of reactive oxygen and nitrogen species leading to more ASN nitration (Figure1).


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Figure 1. Nitration of α-synuclein and its biological consequences. Nitration of ASN results in the formation of ASN aggregates, directly and indirectly induces the cytotoxicity of DA neurons, activates neuroinflammation, and subsequently causes the release of large amounts of reactive oxygen (ROS) and nitrogen species (RNS), potentially leading to more ASN nitration.

3. Nitrated ASN as a potential diagnostic and therapeutic target for PD 3.1 Nitrated ASN as a potential PD biomarker The diagnosis of PD is mainly based on clinical motor symptoms, such as bradykinesia, resting tremor, and rigidity (42). At present, there are no established biomarkers for diagnosing or for following the disease progression of PD, which is a major limitation in clinical practice. ASN, as well as its posttranslational modification products, not only exists in the central nervous system but can also be detected in peripheral tissues (43-45). To explore whether nitrated ASN could be applied as a potential PD biomarker, Prigione examined 25 patients with PD and 30 age- and sex-matched healthy individuals. This study found that the ASN levels in monocytes from PD patients were similar to those in normal controls, whereas the ASN nitration levels were significantly higher and correlated with the levels of reactive oxygen species (46). Xuan Q et al. detected the expression of ASN in colon tissues of different age groups, and they found that nitrated ASN was present in the upper layers of the colon mucosa and submucosa (47). ASN and nitrated ASN levels increased significantly with age. This finding unravelled a correlation between ASN nitration and the degeneration of visceral nerves. As synucleinopathies of the splanchnic nerve are closely related to ageing-induced dyskinesia, the nitration of ASN in the colon tissue of PD patients must be thoroughly scrutinized. ASN has been reported to be highly expressed in erythrocytes (48). Vicente Miranda H et al. examined ASN levels in erythrocytes from 58 PD patients and 30 age-matched healthy individuals and found that the levels of Y39 nitration, Y125 phosphorylation, and glycosylation of ASN in the PD group were significantly elevated compared to those in the control group, while the SUMO modification was reduced. Through the analysis of these posttranslational modifications, a close association between the nitration of ASN and the disease severity of PD patients was revealed (49). This study strongly suggests that nitrated ASN in the blood may become a potential biomarker for PD, although further investigation with larger samples and multi-centre collaborations



is needed.

3.2 Protecting DA neurons through attenuating reactive nitrative stress Given that ASN nitration results in DA neuronal damage, some studies have attempted to protect neurons by developing drugs that reduce nitrative stress for the treatment of PD. Dimethylamine tetracycline has been shown to inhibit the oxidative modification of proteins by blocking the expression of proinflammatory enzymes. This drug was demonstrated to interact with peroxynitrite, although it did not react with superoxide radicals or NO radicals (50). In-depth studies have shown that dimethicone can prevent the tyrosine nitration of ASN, and this effect was stronger than the effect of dimethicone on the inhibition of methionine oxidation. Another study by Schildknecht et al. showed that GKT136901, a NADPH oxidase inhibitor, was a selective scavenger for peroxynitrite. In this study, GKT136901 was shown to prevent tyrosine nitration and dityrosine-dependent dimer formation of ASN (51). Accordingly, Chavarría reported that hydroxyphenyl nitrone inhibited the nitration of ASN and protected SH-SY5Y cells against the toxic effects of 6-OHDA (52). Moreover, hydroxyphenyl nitrone was found to inhibit the inflammatory response by inhibiting the production of nitrite in primary cultures of astrocytes and microglia treated with lipopolysaccharide, a potent inflammatory agent. These results indicate a potential therapeutic use of hydroxyphenyl nitrone against oxygen and nitrogen reactive species. Notably, different animal models of PD must be used to further investigate the efficacy of these drugs. Hydrogen sulfideis associated with the pathogenesis of several neurodegenerative disorders. Yuan YQ et al. discovered a reduction in sulfides in the striatum of the MPTP-induced PD model, along with a reduction in the expression of cystathionine β-synthase (53). They also clarified that virus-transduced overexpression of cystathionine β-synthase in the striatum alleviated the motor deficits and dopaminergic neuron loss in the nigro-striatal pathway. Further mechanistic analysis revealed that this overexpression repressed nitric oxide overproduction and reduced the level of nitrated ASN. Considering the antiapoptotic effect of hydrogen sulfide, Hou et al. investigated the effect of GYY4137, a slow hydrogen sulfide-releasing compound, in the MPTP-induced PD model (54). These researchers found that intraperitoneal injection of


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GYY4137 significantly ameliorated MPTP-caused motor impairments and reduced the loss of dopaminergic neurons in the substantia nigra. Further studies have shown that GYY4137 could reduce ASN nitration in the striatum by relieving nitrative stress. In summary, the nitration of ASN plays a crucial role in the pathogenesis of PD, and a better understanding of the pathological roles of nitrated ASN may provide novel clues to improve the diagnosis and treatment of Parkinson's disease.

Author Information

Corresponding Author *Correspondence to Shengdi Chen, E-mail: [email protected]; or Zhongwang Yu, E-mail: [email protected].

Author Contributions Yixi He, Zhongwang Yu and Shengdi Chen wrote the manuscript and contributed to figure preparation.

Conflict of Interest The authors declare that they have no conflicts of interests.

Funding Sources This work was supported by the National Natural Science Foundation of China (31100765, 81771374, 81430022) and the Natural Science Foundation of Shanghai (11ZR1446600).

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Figure legend: Figure 1. Nitration of α-synuclein and its biological consequences. Nitration of ASN results in the formation of ASN aggregates, directly and indirectly induces the cytotoxicity of DA neurons, activates neuroinflammation, and subsequently causes the release of large amounts of reactive oxygen (ROS) and nitrogen species (RNS), potentially leading to more ASN nitration.



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Alpha-synuclein nitration and its implications in Parkinson's disease

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