Design, synthesis, molecular docking study, and α-glucosidase inhibitory evaluation of novel hydrazide-hydrazone derivatives of 3,4-dihydroxyphenylacetic acid.

A series of novel Schiff base derivatives (1-28) of 3,4-dihydroxyphenylacetic acid were synthesized in a multi-step reaction. All the synthesized Schiff bases were obtained in high yields and their structures were determined by 1HNMR, 13CNMR, and HR-ESI-MS spectroscopy. Except for compounds 22, 26, 27, and 28, all derivatives show excellent to moderate α-glucosidase inhibition. Compounds 5 (IC50 = 12.84 ± 0.52 µM), 4 (IC50 = 13.64 ± 0.58 µM), 12 (IC50 = 15.73 ± 0.71 µM), 13 (IC50 = 16.62 ± 0.47 µM), 15 (IC50 = 17.40 ± 0.74 µM), 3 (IC50 = 18.45 ± 1.21 µM), 7 (IC50 = 19.68 ± 0.82 µM), and 2 (IC50 = 20.35 ± 1.27 µM) shows outstanding inhibition as compared to standard acarbose (IC50 = 873.34 ± 1.67 µM). Furthermore, a docking study was performed to find out the interaction between the enzyme and the most active compounds. With this research work, 3,4-dihydroxyphenylacetic acid Schiff base derivatives have been introduced as a potential class of α-glucosidase inhibitors that have remained elusive till now.

Oxidative Transformations of 3,4-Dihydroxyphenylacetaldehyde Generate Potential Reactive Intermediates as Causative Agents for Its Neurotoxicity.

Neurogenerative diseases, such as Parkinson's disease, are associated, not only with the selective loss of dopamine (DA), but also with the accumulation of reactive catechol-aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is formed as the immediate oxidation product of cytoplasmic DA by monoamine oxidase. DOPAL is well known to exhibit toxic effects on neuronal cells. Both catecholic and aldehyde groups seem to be associated with the neurotoxicity of DOPAL. However, the exact cause of toxicity caused by this compound remains unknown. Since the reactivity of DOPAL could be attributed to its immediate oxidation product, DOPAL-quinone, we examined the potential reactions of this toxic metabolite. The oxidation of DOPAL by mushroom tyrosinase at pH 5.3 produced conventional DOPAL-quinone, but oxidation at pH 7.4 produced the tautomeric quinone-methide, which gave rise to 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxybenzaldehyde as products through a series of reactions. When the oxidation reaction was performed in the presence of ascorbic acid, two additional products were detected, which were tentatively identified as the cyclized products, 5,6-dihydroxybenzofuran and 3,5,6-trihydroxybenzofuran. Physiological concentrations of Cu(II) ions could also cause the oxidation of DOPAL to DOPAL-quinone. DOPAL-quinone exhibited reactivity towards the cysteine residues of serum albumin. DOPAL-oligomer, the oxidation product of DOPAL, exhibited pro-oxidant activity oxidizing GSH to GSSG and producing hydrogen peroxide. These results indicate that DOPAL-quinone generates several toxic compounds that could augment the neurotoxicity of DOPAL.

Screening of Human Gut Bacterial Culture Collection Identifies Species That Biotransform Quercetin into Metabolites with Anticancer Properties.

We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.

The Microbiota-Derived Metabolite of Quercetin, 3,4-Dihydroxyphenylacetic Acid Prevents Malignant Transformation and Mitochondrial Dysfunction Induced by Hemin in Colon Cancer and Normal Colon Epithelia Cell Lines.

Meat diet plays a pivotal role in colorectal cancer (CRC). Hemin, a metabolite of myoglobin, produced after meat intake, has been involved in CRC initiation. The compound, 3,4-dihydroxyphenylacetic acid (3,4HPAA) is a scarcely studied microbiota-derived metabolite of the flavonoid quercetin (QUE), which exert antioxidant properties. The aim of this study was to determine the protective effect of 3,4HPAA against malignant transformation (increased cell proliferation, decreased apoptosis, DNA oxidative damage and augmented reactive oxidative species (ROS) levels) and mitochondrial dysfunction induced by hemin in normal colon epithelial cells and colon cancer cells. The effect of 3,4HPAA was assessed in comparison to its precursor, QUE and to a known CRC protective agent, sulforaphane (SFN). The results showed that both, tumor and normal cells, exposed to hemin, presented increased cell proliferation, decreased caspase 3 activity and cytochrome c release, as well as augmented production of intracellular and mitochondrial ROS. In addition, hemin decreased the mitochondrial membrane potential (MMP) and the activity of complexes I and II of the electron transport chain. These effects of hemin were prevented by the action of 3,4HPAA. The metabolite showed to be more active than QUE and slightly less active than SFN. In conclusion, 3,4HPAA administration could represent a promising strategy for preventing malignant transformation and mitochondrial dysfunction in colon epithelia induced by hemin.

ZIF-67 as a Template Generating and Tuning "Raisin Pudding"-Type Nanozymes with Multiple Enzyme-like Activities: Toward Online Electrochemical Detection of 3,4-Dihydroxyphenylacetic Acid in Living Brains.

Due to its unique structure and high porosity, metal-organic frameworks (MOFs) can act not only as nanozyme materials but also as carriers to encapsulate natural enzymes and thus have received extensive attention in recent years. However, a few research studies have been conducted to investigate MOF as a template to generate and tune nanozymes in the structure and performance. In this work, the "raisin pudding"-type ZIF-67/Cu0.76Co2.24O4 nanospheres (ZIF-67/Cu0.76Co2.24O4 NSs) were obtained by rationally regulating the weight ratio of ZIF-67 and Cu(NO3)2 in the synthesis process. Here, ZIF-67 not only acts as a template but also provides a cobalt source for the synthesis of cobalt copper oxide on the surface of ZIF-67/Cu0.76Co2.24O4 NSs with multiple enzyme-like activities. The ZIF-67/Cu0.76Co2.24O4 NSs can mimic four kinds of enzymes with peroxidase-like, glutathione peroxidase-like, superoxide dismutase-like, and laccase-like activities. Based on its laccase-like activity, an online electrochemical system for continuous monitoring of 3,4-dihydroxyphenylacetic acid with good linearity in the range of 0.5-20 µM and a detection limit of 0.15 µM was established. Furthermore, the alteration of DOPAC in the brain microdialysate before and after ischemia of the rats' brain was also successfully recorded. This work not only raises a new idea for the synthesis of nanozyme materials with multiple enzyme activities but also provides a new solution for the detection of neurotransmitters in living brains.

Antioxidant properties of two novel lipophilic derivatives of hydroxytyrosol.

Two novel lipophilic derivatives of the natural olive oil phenol, hydroxytyrosol (HT), were synthesized using 3,4-dihydroxyphenylacetic acid as starting material. Their antioxidant activities and kinetics compared to HT and TBHQ were assessed by Rancimat, Schaal Oven, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and deep-frying methods. All experiments, including kinetic data analysis based on the Arrhenius equation, utilized in assessing antioxidant activity except the DPPH assay revealed that the new lipophilic HT derivatives exhibited much stronger antioxidant activity than hydroxytyrosol. Tert-butylhydroquinone exhibited stronger antioxidant activity in bulk oil at 65 °C than the new HT derivatives, but showed much lower activity at higher temperatures (>110 °C). This demonstrates that the introduction of bulky alkyl moiety to the ortho-diphenolic structure of HT increased its antioxidant activity. It can be concluded that the new lipophilic HT derivatives satisfy industrial demands for bioactive compounds with strong antioxidant potential at high temperatures.

3,4-Dihydroxyphenylacetaldehyde Is More Efficient than Dopamine in Oligomerizing and Quinonizing α-Synuclein.

Lewy body diseases such as Parkinson's disease involve intraneuronal deposition of the protein α-synuclein (AS) and depletion of nigrostriatal dopamine (DA). Interactions of AS with DA oxidation products may link these neurohistopathologic and neurochemical abnormalities via two potential pathways: spontaneous oxidation of DA to dopamine-quinone and enzymatic oxidation of DA catalyzed by monoamine oxidase to form 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is then oxidized to DOPAL-Q. We compared these two pathways in terms of the ability of DA and DOPAL to modify AS. DOPAL was far more potent than DA both in oligomerizing and forming quinone-protein adducts with (quinonizing) AS. The DOPAL-induced protein modifications were enhanced similarly by pro-oxidation with Cu(II) or tyrosinase and inhibited similarly by antioxidation with N-acetylcysteine. Dopamine oxidation evoked by Cu(II) or tyrosinase did not quinonize AS. In cultured MO3.13 human oligodendrocytes DOPAL resulted in the formation of numerous intracellular quinoproteins that were visualized by near-infrared spectroscopy. We conclude that of the two routes by which oxidation of DA modifies AS and other proteins the route via DOPAL is more prominent. The results support developing experimental therapeutic strategies that might mitigate deleterious modifications of proteins such as AS in Lewy body diseases by targeting DOPAL formation and oxidation. SIGNIFICANCE STATEMENT: Interactions of the protein α-synuclein with products of dopamine oxidation in the neuronal cytoplasm may link two hallmark abnormalities of Parkinson disease: Lewy bodies (which contain abundant AS) and nigrostriatal DA depletion (which produces the characteristic movement disorder). Of the two potential routes by which DA oxidation may alter AS and other proteins, the route via the autotoxic catecholaldehyde 3,4-dihydroxyphenylacetaldehyde is more prominent; the results support experimental therapeutic strategies targeting DOPAL formation and DOPAL-induced protein modifications.

Protocatechuic and 3,4-Dihydroxyphenylacetic Acids Inhibit Protein Glycation by Binding Lysine through a Metal-Catalyzed Oxidative Mechanism.

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 µmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 µmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA-phenolic acid adducts was estimated by nanoflow liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA-phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.

Enzyme-Free Plasmonic Biosensor for Direct Detection of Neurotransmitter Dopamine from Whole Blood.

Complex biological fluids without pretreatment, separation, or purification impose stringent limitations on the practical deployment of label-free plasmonic biosensors for advanced assays needed in point of care applications. In this work, we present an enzyme-free plasmonic neurotransmitter dopamine biosensor integrated with a microfluidic plasma separator. This integrated device allows the in-line separation of plasma directly from the bloodstream and channels it to the active detection area, where inorganic cerium oxide nanoparticles function as local selective dopamine binding sites through strong surface redox reaction. A thorough understanding and engineering of the nanoparticles is carried out to maximize its dopamine sensitivity and selectivity. We obtain detection of dopamine at 100 fM concentration in simulated body fluid and 1 nM directly from blood without any prior sample preparation. The detection selectivity is found to be at least five-times higher compared to the common interfering species. This demonstration shows the feasibility of the practical implementation of the proposed plasmonic system in detection of variety of biomarkers directly from the complex biological fluids.

Formation of large oligomers of DOPAL-modified α-synuclein is modulated by the oxidation of methionine residues located at C-terminal domain.

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

Study of Vesicular Monoamine Transporter 2 in Myopic Retina Using [18F]FP-(+)-DTBZ.

PURPOSE: To investigate the relationship between expression level of vesicular monoamine transporter 2 (VMAT2) and myopia, as well as the feasibility of noninvasive myopia diagnosis through imaging VMAT2 in retina by using [18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-(+)-DTBZ). PROCEDURES: The right eyes of ten guinea pigs were deprived of vision to establish form-deprived (FD) myopia and the left eyes were untreated as the self-control eyes. The location and expression level of VMAT2 in the eyes were detected by micro-positron emission tomography (PET)/X-ray computed tomography (CT) imaging through using [18F]FP-(+)-DTBZ. Immunofluorescence staining and Western blot were used to confirm the location and expression level of VMAT2 in the eyes. The concentrations of dopamine (DA) and its metabolites including 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were also investigated by high-performance liquid chromatography. RESULTS: The right eyes deprived of vision were obviously myopic (- 3.17 ± 1.33 D) after procedure, while the left eyes were hyperopic (4.60 ± 0.83 D, P < 0.0001). The main expressions of VMAT2 in the eyes were located in retina. VMAT2 was significantly reduced in the myopic retina compared to the normal one from PET/CT results (P = 0.0008), which could also be verified by Western blots (P = 0.029). The concentrations of DA, DOPAC, and HVA in the FD eyes were all significantly less than those in the control eyes (P = 0.024, P = 0.018, P = 0.008). As a role of storing and releasing DA in vesicles, VMAT2 was demonstrated positively correlating with the amounts of DA (P = 0.030), DOPAC (P = 0.038), and HVA (P = 0.025) through Pearson's correlation coefficient test. CONCLUSIONS: We demonstrate that [18F]FP-(+)-DTBZ can be used to noninvasively image VMAT2 in retina. The expression level of VMAT2 in retina may act as a new biomarker for myopia diagnosis. The decreasing of VMAT2 expression level may play an important role in the development of myopia through correspondingly reducing the amount of DA in retina.

Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of α-Synuclein.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic and reactive product of dopamine catabolism. In the catecholaldehyde hypothesis for Parkinson's disease, it is a critical driver of the selective loss of dopaminergic neurons that characterizes the disease. DOPAL also cross-links α-synuclein, the main component of Lewy bodies, which are a pathological hallmark of the disease. We previously described the initial adduct formed in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine (DCPL). Here, we examine the chemical basis for DOPAL-based cross-linking. We find that autoxidation of DCPL's catechol rings spurs its decomposition, yielding an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings to give an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. This product is formed by an intermolecular carbon-carbon bond between the isoindole rings of the two DCILs that generates two structurally nonequivalent and separable atropisomers. Using α-synuclein, we demonstrate that the DOPAL-catalyzed formation of oligomers can be separated into two steps. The initial adduct formation occurs robustly within an hour, with DCPL as the main product, and the second step cross-links α-synuclein molecules. Exploiting this two-stage reaction, we use an isotopic labeling approach to show the predominant cross-linking mechanism is an interadduct reaction. Finally, we confirm that a mass consistent with a di-DCIL linkage can be observed in dimeric α-synuclein by mass spectrometry. Our work elucidates previously unknown pathways of catechol-based oxidative protein damage and will facilitate efforts to detect DOPAL-based cross-links in disease-state neurons.

A novel and efficient synthesis of 3,4-dihydroxyphenylacetic ester and amide derivatives/conjugates and assessment of their antioxidant activity.

Phenolic acids, a sub-class of polyphenols, are widely studied. By contrary, 3,4-dihydroxyphenylacetic acid is scarcely studied. For this purpose, a series of 3,4-dihydroxyphenylacetic acid ester and amide derivatives/conjugates were synthesised for the first time. A systematic study has been performed to quantitatively identify the functional groups present in these compounds using different techniques such as 1H NMR, 13C NMR and ESI MS. The synthesised compounds were evaluated for their in vitro antioxidant activity by a DPPH radical-scavenging assay. Their physico-chemical profile is also studied using Molinspiration tool. Among all tested compounds, amidoester 36 showed the best scavenging activity possessing an EC50 17 µΜ and improved physico-chemical properties compared to the parent compound.

Facile preparation of core cross-linked nanomicelles based on graft copolymers with pH responsivity and reduction sensitivity for doxorubicin delivery.

To achieve passive targeting and controlled drug release at tumor sites trigged by low pH value and high level of glutathione (GSH), optimized delivery system for doxorubicin (DOX) based on core cross-linked nanomicelles was developed in this research. Particularly, methoxypoly(ethylene glycol)-nitrophenyl carbonate (mPEG-NPC) and 3,4-dihydroxyphenylaceticacid were grafted onto synthesized poly(N,N'-cystamine bisacrylamide-N-Boc-1,2-diaminoethane) (poly(CBA-DAE)) to give methoxypoly(ethylene glycol)-g-poly(N,N'-cystamine bisacrylamide-N-Boc-1, 2-diaminoethane)-g-3, 4-dihydroxyphenylaceticacid (mPEG-g-SS-PCD-DA). Core cross-linked micelles (CCLMs/SS) with a decreased average particle size of 121nm were prepared by adding Fe3+ into uncross-linked micelles (UCLMs/SS) self-assembled from mPEG-g-SS-PCD-DA. DOX-loaded CCLMs/SS exhibited minimal drug leakage (17.3%) under simulated blood conditions compared to DOX-loaded UCLMs/SS (31.3%). Fast drug release (52.4%) of DOX-loaded CCLMs/SS was achieved compared to DOX-loaded CCLMs/CC (32.9%) without disulfide bonds under simulated lysosomes condition over 42h. The cytotoxicity of DOX-loaded CCLMs/SS against A549 cells pretreated with 40mM NH4Cl was decreased significantly compared to that without NH4Cl treatment, and it is higher than that of DOX-loaded CCLMs/CC, further confirmed DOX release was triggered by the low pH value and high level of reductive agents of lysosomes. Compared with free DOX, DOX-loaded CCLMs/SS showed enhanced cellular uptake ability during 24h of incubation through endocytosis. Besides, charge conversion of micelles happened when pH varied from 7.4 to 6.5, which facilitates the cellular uptake against A549 cells. In summary, all these results indicated that CCLMs/SS as a new type of intelligent nanocarriers exhibited excellent potential for drug delivery.

Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding.

Impairment of growth hormone (GH) signaling has been associated with increased feeding and adiposity. The gastric hormone ghrelin, in addition to its GH-secretagogue effects, stimulates food intake after both central and peripheral administration. In the present study we further investigated the feeding regulatory role of the ghrelin-GH axis in a mouse model of isolated GH deficiency due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. We evaluated the effects of intracerebroventricular ghrelin administration on feeding behavior, related hypothalamic neuropeptides and neurotransmitters, and serum ghrelin levels in mice homozygous for GHRHKO allele (-/-) and heterozygous (+/-) control animals. Vehicle-treated GHRHKO mice showed increased food intake compared to heterozygotes, associated with increased circulating ghrelin levels. Moreover, -/- mice showed elevated hypothalamic levels of neuropeptide Y (NPY), agouti-related peptide (AgRP) mRNAs and norepinephrine (NE) and decreased corticotropin-releasing hormone (CRH) mRNA levels. Ghrelin treatment significantly augmented food intake in both genotypes, but the relative increase compared to vehicle-treated animals was higher in -/- than +/- mice. In the hypothalamus, ghrelin increased AgRP and decreased CRH gene expression only in heterozygous mice, while it induced a significant reduction in proopiomelanocortin (POMC) mRNA levels in -/- mice. Ghrelin treatment also decreased hypothalamic serotonin (5-hydroxytriptamine, 5-HT) and dopamine (DA) levels in both genotypes. Additionally, we observed increased DA metabolism induced by ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice could lead to increased feeding secondary to elevated circulating levels of ghrelin, and the obesogenic phenotype is likely mediated by elevated NPY and AgRP, and decreased CRH gene expression in the hypothalamus.

Superoxide is the critical driver of DOPAL autoxidation, lysyl adduct formation, and crosslinking of α-synuclein.

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomerize α-synuclein, the main component of protein aggregates in Lewy bodies. In this work we examine the connection between reactive oxygen species and DOPAL autoxidation. We show that superoxide propagates a chain reaction oxidation, and that this reaction is dramatically inhibited by superoxide dismutase. Moreover, superoxide dismutase prevents DOPAL from forming dicatechol pyrrole adducts with lysine and from covalently crosslinking α-synuclein. Given that superoxide is a major radical byproduct of impaired cellular respiration, our results provide a possible mechanistic link between mitochondrial dysfunction and synuclein aggregation in dopaminergic neurons.

Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of α-Synuclein.

Parkinson's disease has long been known to involve the loss of dopaminergic neurons in the substantia nigra and the coincidental appearance of Lewy bodies containing oligomerized forms of α-synuclein. The "catecholaldehyde hypothesis" posits a causal link between these two central pathologies mediated by 3,4-dihydroxyphenylacetaldehyde (DOPAL), the most toxic dopamine metabolite. Here we determine the structure of the dominant product in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine adduct. This novel modification results from the addition of two DOPAL molecules to the Lys sidechain amine through their aldehyde moieties and the formation of a new carbon-carbon bond between their alkyl chains to generate a pyrrole ring. The product is detectable at low concentrations of DOPAL and its discovery should provide a valuable chemical basis for future studies of DOPAL-induced crosslinking of α-synuclein.

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme-substrate interactions.

BACKGROUND: Extradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step. METHODS: This study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments. RESULTS: The equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2×10(4) to 1×10(6), suggesting there are distinctive differences in how the enzyme-substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts. CONCLUSIONS: Condition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme-substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity. GENERAL SIGNIFICANCE: Substrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation.

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL).

Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.

The serotonin aldehyde, 5-HIAL, oligomerizes alpha-synuclein.

In Parkinson's disease (PD) alpha-synuclein oligomers are thought to be pathogenic, and 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate aldehyde intermediate in neuronal dopamine metabolism, potently oligomerizes alpha-synuclein. PD involves alpha-synuclein deposition in brainstem raphe nuclei; however, whether 5-hydroxyindoleacetaldehyde (5-HIAL), the aldehyde of serotonin, oligomerizes alpha-synuclein has been unknown. In this study we tested whether 5-HIAL oligomerizes alpha-synuclein in vitro and in PC12 cells conditionally over-expressing alpha-synuclein. Alpha-synuclein oligomers were quantified by western blotting after incubation of alpha-synuclein with serotonin and monoamine oxidase-A (MAO-A) to generate 5-HIAL or dopamine to generate DOPAL. Oligomerization of alpha-synuclein in PC12 cells over-expressing the protein was compared between vehicle-treated cells and cells incubated with levodopa to generate DOPAL or 5-hydroxytryptophan to generate 5-HIAL. Monoamine aldehyde mediation of the oligomerization was assessed using the MAO inhibitor, pargyline. Dopamine and serotonin incubated with MAO-A both strongly oligomerized alpha-synuclein (more than 10 times control); pargyline blocked the oligomerization. In synuclein overexpressing PC12 cells, levodopa and 5-hydroxytryptophan elicited pargyline-sensitive alpha-synuclein oligomerization. 5-HIAL oligomerizes alpha-synuclein both in vitro and in synuclein-overexpressing PC12 cells, in a manner similar to DOPAL. The findings may help explain loss of serotonergic neurons in PD.