Dopamine-Derived Cotoxins and the Unique Vulnerability of Dopaminergic Neurons in Parkinson’s Disease

Dopamine-Derived Cotoxins and the Unique Vulnerability of Dopaminergic Neurons in Parkinson’s Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dopamine-Derived Cotoxins and the Unique Vulnerability of Dopaminergic Neurons in Parkinson’s Disease Praneet Kaur Marwah, Lin Rayes, Sanjana Aiella, Alexa Atty, Ali Rashid, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9280779/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The fundamental problem in Parkinson’s disease is the selective death of dopaminergic neurons in the substantia nigra. This selective death is facilitated by products of dopamine oxidation including a toxin, DHBT-1 (7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2 H -benzo[b][1,4]thiazine-3-carboxylic acid) and a cotoxin, BT-2 (7-(2-aminoethyl)- 2H -benzo[b][1,4]thiazin-5-ol). The cytotoxicity of these compounds was tested both individually and in the presence of the mitochondrial complex I inhibitors rotenone and Paraquat, both of which are implicated as causes of Parkinson’s disease. BT-2 potentiates the toxicities of these inhibitors. It also amplifies the toxicity of DHBT-1, reported to also be a complex I inhibitor. Although BT-2 is somewhat unstable, an analogue (2,2-dimethyl-BT-2) is much more stable than BT-2 itself and also enhances the toxicities of DHBT-1, rotenone and Paraquat. We suggest that DHBT-1 and BT-2 are normally detoxified by polymerization into neuromelanin but may accumulate under certain conditions and contribute, in concert with other stresses, to the selective death of dopaminergic neurons. Biological sciences/Drug discovery Biological sciences/Neuroscience Benzothiazine Cysteinyl-dopamine Dopamine Neuromelanin Paraquat Parkinson’s disease Rotenone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The movement disorders characteristic of Parkinson’s disease result from the death of dopaminergic neurons in the substantia nigra. Many factors contributing to the disease or its animal models have been identified. These include mutations in proteins such as α-synuclein, Parkin and PINK1 1-4 and mitochondrial inhibitors such as rotenone and Paraquat. 5-7 The mutant proteins are expressed not just in dopaminergic neurons, however, and the mitochondrial inhibitors should affect nearly all cells in the body. Although Parkinson’s disease has been studied intensely, a central question remains: Why are dopaminergic neurons uniquely vulnerable to these stresses? 8-11 The extensive arborization of dopaminergic neurons and the attendant stress of dispersed bioenergetic and synaptic functions has been suggested. 8,9 There is also considerable evidence, however, that dopamine degradation products play a crucial role. 12-16 Transgenic mice overexpressing the dopamine transporter in dopamine neurons specifically display a loss of midbrain dopamine neurons and motor deficits reversed by L-DOPA. This is associated with an increase in the dopamine oxidation product, cysteinyl-dopamine. 12 Knockdown of the vesicular monoamine transporter (VMAT2) in the substantia nigra of rats causes an increase in cytosolic dopamine followed by degeneration of nigrostriatal dopaminergic neurons, deficits in dopamine-mediated behaviors, and the formation of aberrant α-synuclein. 13 On the cellular level, treating SH-SY5Y cells with dopamine oxidized using tyrosinase results in mitochondrial membrane depolarization, reduction of ATP synthesis, opening of the mitochondrial transition pore and ultimately cell death. 16 Primary cultures of mouse cortical neurons are killed by cysteinyl-dopamine and/or its breakdown products. 17 PC-12 cells are killed by a cytotoxic preparation obtained when cysteinyl-dopamine is treated with sodium hypochlorite. 18 Scavenging hypochlorous acid protects against the cytotoxicity of cysteinyl-dopamine but not against hypochlorous acid-treated cysteinyl-dopamine (HOCD), suggesting that the cytoxic activity is downstream of cysteinyl-dopamine. Despite this evidence, there have been relatively few studies attempting to identify specific dopamine-derived toxins and their mechanism of action. Dryhurst’s group identified 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2 H -benzo[b][1,4]thiazine-3-carboxylic acid or DHBT-1 as the major oxidation product of cysteinyl-dopamine. 19-21 They also showed that it is an inhibitor of complex I of the mitochondrial respiratory chain but did not assess cytotoxicity. 21,22 The same group identified 7-(2-aminoethyl)- 2H -benzo[b][1,4]thiazin-5-ol or BT-2 but found it too unstable to pursue. 22,23 In fact, significant obstacles to studying these compounds may be that they are unstable and tend to polymerize and that current methodology for preparing them is laborious and requires specialized chemical facilities. Here we present rapid and simple procedures for preparing and purifying DHBT-1 and BT-2. We also describe the synthesis of 2,2-dimethyl-BT-2, a very stable analogue of BT-2. Using these compounds, we test the hypothesis that they increase the toxicity of mitochondrial complex I inhibitors and account for the unique vulnerability of dopaminergic neurons to stresses implicated in Parkinson’s disease. Results Cysteinyl-dopamine is a primary oxidation product of dopamine in vivo and a precursor of neuromelanin (Fig. 1 ). Previously, we showed that a cytotoxic preparation is obtained when dopamine is oxidized by tyrosinase in the presence of cysteine, and the resulting cysteinyl-dopamine is then treated with sodium hypochlorite. 18 To identify and characterize the active compounds in this preparation, we scaled up the synthesis of cysteinyl-dopamine by oxidizing dopamine using ceric ammonium nitrate instead of tyrosinase. 24 This preparation of cysteinyl-dopamine confirms that cysteinyl-dopamine and hypochlorous acid-oxidized cysteinyl-dopamine (HOCD) are cytotoxic to PC12 cells (Fig. 2 A). Upon purification, the major component of HOCD was determined to be DHBT-1, a dihydrobenzothiazine previously recognized by others as a major product of cysteinyl-dopamine oxidized by different methods. 19,25 DHBT-1, identified by its absorption spectrum and confirmed by high-resolution mass spectrometry, is obtained from cysteinyl-dopamine with very high yield. DHBT-1 kills PC12 cells; however, it is much less cytotoxic than either cysteinyl-dopamine or HOCD (Fig. 2 A). The loss of toxicity upon purification of DHBT-1 suggested the existence of a cotoxin augmenting DHBT-1 toxicity. In fact, when DHBT-1 is purified by reverse-phase chromatography, a cotoxin activity elutes after DHBT-1. 26 High resolution mass spectrometry identified the chemical formula of the cotoxin as C 10 H 12 N 2 OS consistent with BT-2. Although BT-2 can be prepared by oxidizing cysteinyl-dopamine with sodium hypochlorite, Napolitano et al. reported an efficient synthesis of the cysteinyl-L-DOPA analogue using potassium ferricyanide as the oxidizing agent at 37°C. 27 Using this procedure, we prepared BT-2 at a low concentration (~ 2 mM) to minimize polymerization of the product. This preparation of BT-2 is not very toxic itself, but it greatly increases the toxicity of DHBT-1 (Fig. 2 B). The fact that BT-2 enhances the lethality of DHBT-1 raises the possibility that it might enhance the toxicity of other factors implicated in Parkinson’s disease, thereby contributing to the unique vulnerability of dopaminergic neurons to these factors. Because DHBT-1 has been reported to inhibit complex I of the mitochondrial respiratory chain, 21,22 we tested BT-2 to see if it potentiates the toxicity of other complex I inhibitors. Indeed, rotenone and Paraquat are quite toxic in the presence of BT-2 even at concentrations at which they otherwise have negligible effect (Fig. 3 A and B). To see whether this cotoxicity is unique to complex I inhibitors or applies to any stress, we also tested the effect of BT-2 on the toxicity of the complex III inhibitor antimycin A and the ATP synthase inhibitor oligomycin. BT-2 increases the toxicity of these compounds, but only at concentrations that already cause significant cell death (Fig. 3 C and D). As reported by others, 19,28 BT-2 is not very stable. To analyze its biological properties, we synthesized, purified and tested the cytotoxicity of the compound on the same day. One reason for the instability of BT-2 is its tendency to dimerize at the C2 position especially at acidic pH (Fig. 4 A). While BT-2 itself absorbs in the ultraviolet (Fig. 4 B), it decomposes to a purple product with an absorbance maximum at 580 nm (Fig. 4 C) consistent with formation of decarboxylated trichochrome F. 23,27 In 0.5 M HCl, BT-2 converts fairly rapidly to this trichochrome, but DHBT-1 does not (Fig. 4 D). A stable analogue of BT-2 may be synthesized by using using penicillamine instead of cysteine. This adds two methyl groups at the C2 position of BT-2 yielding 7-(2-aminoethyl)- 2,2-dimethyl- 2H -benzo[b][1,4]thiazin-5-ol (Me 2 BT-2). The absorption spectrum of Me 2 BT-2 is very similar to that of BT-2 (Fig. 4 B), and both have a higher absorbance at 200 nm than DHBT-1. A significant difference is that Me 2 BT-2 does not convert to the trichochrome in 0.5 M HCl (Fig. 4 D). In fact, Me 2 BT-2 is quite stable and can be kept in aqueous solution for several weeks at 4°C. Moreover, because Me 2 BT-2 is stable under acidic conditions, it can be synthesized in high yield in one step by oxidizing dopamine in the presence of penicillamine in HCl. Like BT-2, Me 2 BT-2 potentiates the toxicity of DHBT-1 (Fig. 5 A) and rotenone (Fig. 5 B). Given that BT-2 and Me 2 BT-2 both potentiate the toxicity of rotenone, one might ask whether DHBT-1 also exhibits this cotoxin activity. DHBT-1 does not increase the toxicity of rotenone, however, (Fig. 5 C), so DHBT-1 is a toxin but not a cotoxin. When tested with other mitochondrial inhibitors, Me 2 BT-2 shows the same pattern of cotoxicity as BT-2. Like BT-2, it amplifies the toxicities of rotenone (Fig. 5 B) and Paraquat (Fig. 6 A), but it has less effect on the toxicity of antimycin A (Fig. 6 B) and no effect with oligomycin (Fig. 6 C). Interestingly, the toxicity of DHBT-1 depends on the medium. It is toxic at a significantly lower concentration when PC12 cells are treated in DMEM/F12 medium than when they are in Ham’s F12K medium (Fig. 7 A). Because Me 2 BT-2 is stable at higher concentrations than BT-2, its toxicity can be measured. It is actually toxic at somewhat lower concentrations than DHBT-1, but its toxicity does not depend on the medium in the same way (Fig. 7 B). Furthermore, the cotoxin activity of Me 2 BT-2 is more evident when tested in Ham’s F12K medium than in DMEM/F12 medium (Fig. 7 C). The same seems to be true of BT-2. Therefore, cotoxicity of Me 2 BT-2 and BT-2 and toxicity of DHBT-1 are both affected by the composition of the medium, but in different ways. Discussion Hallmark characteristics of Parkinson’s disease include the loss of dopaminergic neurons in the substantia nigra, motor deficits caused by the impairment of this motor circuit, and the appearance of α-synuclein deposits in Lewy bodies. A number of reports suggest that dopamine oxidation and mitochondrial dysfunction play integral roles in this neurodegeneration. 5,12–14,29,30 As mentioned, cytoplasmic oxidation of dopamine may be increased by overexpressing the cell membrane dopamine transporter or by knocking down the vesicular monoamine transporter. Both of these lead to loss of dopaminergic neurons, development of motor deficits and an increase in α-synuclein. 12,13 Similarly, disruption of mitochondrial respiration by chronic treatment with rotenone creates a rat model of Parkinson’s disease in which Lewy bodies are formed, movement is impaired, and dopaminergic neurons are lost. 5 Mitochondrial dysfunction caused by a DJ-1 loss-of-function mutation leads to dopamine oxidation and α-synuclein accumulation. 14 The identity of the dopamine oxidation products mediating this degeneration is still a matter of debate. The ultimate product of dopamine oxidation is neuromelanin, the black pigment that gives the substantia nigra its name. Neuromelanin is an amorphous polymer incorporating products of dopamine and cysteinyl-dopamine. Its biosynthesis parallels that of pheomelanin except that the latter is made from L-DOPA and cysteine. The initial steps in the biosynthesis of neuromelanin (Fig. 1 ) are well known, and the first intermediates are cysteinyl-dopamine, DHBT-1 and BT-2. 20,25,31 The biological effects of these intermediates have not received much attention, however, a significant obstacle being that they naturally polymerize especially at high concentrations. For this reason, it is difficult to synthesize, purify and store these compounds, especially BT-2, in a form suitable for cytoxicity studies. Dopamine oxidizes slowly but spontaneously in the presence of O 2 to form the dopamine quinone. The dopamine quinone may cyclize and oxidize to form aminochrome, or it may react with thiols such as cysteine to form cysteinyl-dopamine. The reaction with cysteine is quite fast, and the product, cysteinyl-dopamine, is relatively stable. In contrast, aminochrome decomposes rather quickly. Consistent with this, cysteinyl-dopamine is found in the brain 32–34 but the detection of aminochrome has not been reported. Although cysteinyl-dopamine kills cells, 17 we have argued that this toxicity is attributable to its conversion to other products by endogenous oxidants such as hypochlorous acid. 18 Taurine, which scavenges hypochlorous acid, protects against the cytotoxicity of cysteinyl-dopamine but not against HOCD, cysteinyl-dopamine already oxidized with hypochlorous acid. The primary products of cysteinyl-dopamine oxidation are DHBT-1 and BT-2. These are alternative products: less oxidation favors DHBT-1; more oxidation yields BT-2. Studying melanin biosynthesis, Napolitano et al. demonstrated this dichotomy using L-DOPA instead of dopamine with ferricyanide as the oxidizer. 27 To make DHBT-1, an oxidizer is required only to initiate oxidation of cysteinyl-dopamine, thereby forming a catalytic amount of o-quinone imine. The reduction potentials are such that the o-quinone imine will then oxidize cysteinyl-dopamine and itself be reduced to DHBT-1 (Fig. 1 ). This cycling results in complete conversion of cysteinyl-dopamine to DHBT-1. More oxidizer actually lowers the yield of DHBT-1 because it oxidizes more cysteinyl-dopamine making it unavailable to reduce the o-quinone imine to DHBT-1. Higher concentrations of oxidizer increase the formation of BT-2, because greater oxidation of cysteinyl-dopamine forces the o-quinone imine to to take the less favored pathway leading to BT-2 (Fig. 1 ). BT-2 oxidizes readily, so it is unstable and decomposes over a period of hours. It may dimerize to form a trichochrome or react with other thiols like cysteine or glutathione. Our synthesis of BT-2 uses a lower concentration of cysteinyl-dopamine, because the lower concentration of product slows dimerization and improves yield and stability of BT-2. It also limits the amount that may be synthesized, however, and the concentration of stock solutions. The oxidation of BT-2 may be prevented by adding methyl groups to the C2 carbon. Napolitano et al. took advantage of this approach, using D-penicillamine instead of cysteine to produce more a stable product with L-DOPA. 28 They were interested in characterizing intermediates in the formation of melanin, however, and not in their biological activities. Interestingly, the dimethylated analogue of BT-2 (Me 2 BT-2) can be synthesized from penicillamine and dopamine in one step using a slightly higher concentration of oxidant (ceric ammonium nitrate) and lower concentration of thiol (penicillamine). Dopamine oxidation then leads to formation of penicillaminyl-dopamine, and the latter is further oxidized in HCl to Me 2 BT-2. Because Me 2 BT-2 cannot dimerize to a trichochrome, the reaction stops there. BT-2 cannot be made in the same way, because it goes on to dimerize and form other products. Having the capacity to make DHBT-1, BT-2 and the cotoxin Me 2 BT-2, it is now possible to test their involvement in Parkinson’s disease. Are these toxins present in dopaminergic neurons at concentrations physiologically relevant to Parkinson’s disease? We found that DHBT-1 is toxic at millimolar concentrations (Fig. 2 A), while BT-2 acts as a cotoxin at 100 µM (Fig. 2 B, 3 A and 3 B). While these concentrations may not seem especially potent, several issues should be considered. First, most people do not get Parkinson’s disease, suggesting that these compounds normally polymerize to neuromelanin and do not reach toxic concentrations. Second, we observed toxicity in a majority of cells after two days of treatment. Parkinson’s disease kills dopaminergic neurons over decades, so these compounds may cause damage that accumulates over time. Alternatively, toxicity may occur episodically with these toxins reaching higher concentrations transiently and in specific locations. In addition, the toxic dose seems to depend on conditions (Fig. 7 ), so there may be factors that render some individuals more susceptible to these toxins. Finally, the cotoxin is synergistic and is only needed to augment the toxicity of other stresses. Therefore, important questions are: 1) Are there conditions or events that increase the concentrations or toxicities of DHBT-1 and BT-2 in some people? 2) How do these compounds synergize with other factors linked to Parkinson’s disease to cause neurotoxicity? 3) How does BT-2 cause cotoxicity and might this identify potential targets for therapeutic intervention? Idiopathic cases of Parkinson’s disease may involve a diminished ability to clear DHBT-1 and BT-2 or an increased sensitivity to their toxicity. Understanding this connection could be significant both as a diagnostic indicator of susceptibility to Parkinson’s disease and for the identification of potential therapeutic targets. While we do not yet know how conditions affect the concentrations of these toxins or their interactions with their targets, this clearly requires investigation. A significant unanswered question is why are dopaminergic neurons exceptionally vulnerable to other factors linked to Parkinson’s disease including aggregates of α-synuclein and mutations in proteins such as Parkin, PINK1 and LRRK2. If the cotoxin BT-2 potentiates the toxicities of these factors, then it could be the missing piece that answers this question. The availability of the stable BT-2 analogue, Me 2 BT-2, now makes if more practical to test the effect of the cotoxin on the toxicities of these other factors. Finally, how does BT-2 cause cotoxicity? Knowing how the cotoxin acts will not only elucidate the mechanism of Parkinson’s disease, but it may identify potential therapeutic interventions. To date, cell culture has not provided much information about the molecular processes leading to the death of dopaminergic neurons, perhaps because studies have not included the dopamine-derived toxins DHBT-1 and BT-2 but have relied instead on artificial dopaminergic toxins like 6-hydroxydopamine or 1-methyl-4-phenylpyridinium (MPP+). The availability of Me 2 BT-2 will make it more practical for researchers to use cell culture rather than animals to investigate the interaction of cotoxin with chemicals and mutations implicated in Parkinson’s disease. This in turn may provide an efficient, high-throughput, cell-culture screen for treatments that block the toxicity of these Parkinsons’s agents and thereby have the potential to slow or stop the progression of the disease. In conclusion, we have shown that BT-2 is a cotoxin that potentiates the toxicities of mitochondrial inhibitors including rotenone, Paraquat and the endogenous toxin DHBT-1. Thus, BT-2 and DHBT-1 may be missing pieces in the puzzle of Parkinson’s disease, and knowing how they fit into the processes leading to the death of dopaminergic neurons may lead to a more complete understanding of the progression of the disease and better-informed design of interventions to slow or stop it. Methods Materials Sodium hypochlorite, ceric ammonium nitrate, dopamine HCl, L-cysteine, and D-penicillamine were purchased from Sigma/Aldrich, as were rotenone, antimycin A, methyl viologen (Paraquat) and oligomycin. Chemical Syntheses Cysteinyl dopamine : Ten ml of ceric ammonium nitrate (0.4 M in 0.25 N HCl) was added to 5 ml of dopamine (0.4 M in 0.25 N HCl). This was immediately added to 10 ml of L-cysteine (0.24 M in 0.25 N HCl), and the mixture was applied to a Sep-Pak 35 cc C18 Vac cartridge and eluted with 52 ml of 0.1 M HCl. Cysteinyl dopamine was identified and quantified by its UV absorption spectrum (extinction coefficients: 3,810 M −1 cm − 1 at 254 nm and 2,860 M −1 cm − 1 at 293 nm). 24 Yield averaged about 45%. DHBT-1 : Cysteinyl-dopamine (0.4 mmol) was mixed with 35 ml of 50 mM potassium phosphate, pH 6.8. Six percent NaOCl (0.5 ml or 0.4 mmol) was added. The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 26 ml of H 2 O followed by 52 ml of 10% ethanol. DHBT-1 was identified and quantified by its UV absorption spectrum (extinction coefficients: 11,480 M −1 cm − 1 at 234 nm and 1,905 M −1 cm − 1 at 307 nm). 19 Yield was close to 100%. Identification was confirmed by high resolution mass spectrometry (Supplementary Fig. 1) which gave M + z = 255.0795 (theoretical = 255.0803) consistent with a chemical formula of C 11 H 14 N 2 O 3 S). Me 2 BT-2 Ten ml of ceric ammonium nitrate (0.42 M in 1 N HCl) was added to 5 ml of dopamine (0.4 M in 1 N HCl). This was immediately added to 10 ml of D-penicillamine (0.2 M in 1 N HCl). The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 39 ml of 4% ethanol followed by 72 ml of 10% ethanol. The UV absorption spectrum in H 2 O has maxima at 232 nm (ϵ = 17,400 M −1 cm − 1 ) and 302 nm (ϵ = 2,060 M −1 cm − 1 ). Yield was about 30%. Identification was confirmed by high resolution mass spectrometry (Supplementary Fig. 2) which gave M + z = 237.1053 (theoretical = 237.1062) consistent with a chemical formula of C 12 H 16 N 2 OS). BT-2 Cysteinyl-dopamine (0.08 mmol) was mixed with 40 ml of 50 mM potassium phosphate, pH 6.8 at 37°C. Then 3.6 ml of 100 mM potassium ferricyanide (0.36 mmol) was added at 37°C with stirring. The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 26 ml of H 2 O followed by 52 ml of 21% ethanol. BT-2 was identified by its UV absorption spectrum (maxima at 225 nm and 300 nm). Because BT-2 is unstable on drying, its concentration was estimated using the same extinction coefficient as its analogue Me 2 BT-2 (ϵ = 17,400 M −1 cm − 1 at 225 nm). Yield was about 7%. For toxicity experiments, BT-2 was added to cell cultures immediately following synthesis and purification. Spectrometry Concentrations of products were determined from UV absorption spectra recorded on a Molecular Devices SpectraMax M2 operated in cuvette mode. Spectra were recorded in H 2 O, and concentrations calculated using the extinction coefficients listed above. High resolution mass spectrometry was performed on a Thermo Scientific LTQ Orbitrap XL spectrometer in the Lumigen Instrument Center. Cytotoxicity PC12 cells (ATCC Cat# CRL-1721.1, RRID: CVCL F659) were used to test cytotoxicity because they are an adrenergic cell line and are commonly used as a model for Parkinson’s disease studies. 35 Furthermore, we used them earlier to demonstrate the toxicity of hypochlorite-oxidized cysteinyl-dopamine and its synergy with rotenone. 18 PC12 cells (were cultured in 6-well plates in 2 ml of Ham’s F-12K (Kaighn’s) medium, supplemented with 15% heat-inactivated horse serum, 2.5% fetal bovine serum and 1% penicillin/streptomycin/ glutamine at 37°C in a 5% CO 2 atmosphere. Toxins were added when cells reached confluence. After 48 hours of treatment, cell viability was measured using the trypan blue exclusion assay. Cells were detached using 0.1% trypsin, centrifuged, and resuspended in 0.2% trypan blue in Hanks Buffered Salt Solution. After 3 min, live and dead cells were counted using a Countess Automated Cell counter (Invitrogen). Statistical analysis For experiments testing cotoxicity (Figs. 2 B, 3 , 5 and 6 ), the statistical significance of the difference between slopes of lines with or without cotoxin was determined using GraphPad Prism. This calculates a two-tailed p value testing the null hypothesis that the slopes are identical. Significance is indicated as ns (p ≥ 0.05), *(p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001). Abbreviations BT-2, 7-(2-aminoethyl)- 2H -benzo[b][1,4]thiazin-5-ol; DHBT-1, 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2 H -benzo[b][1,4]thiazine-3-carboxylic acid; Me 2 BT-2, 7-(2-aminoethyl)-2,2-dimethyl- 2H -benzo[b][1,4]thiazin-5-ol; CysDA, Cysteinyl-dopamine; HOCD, hypochlorite-oxidized cysteinyl-dopamine. Declarations Competing interests: All authors declare no financial or non-financial competing interests. Author Contribution P.K.M designed experiments and acquired, analyzed, and interpreted data. L.R. prepared compounds and conducted toxicity experiments. S.A., A.A., A.R. and K.S. prepared compounds and performed experiments. E.P. contributed to development of chemical synthesis procedures and performed chemical analysis. D.N. designed and supervised chemical syntheses and cytotoxicity experiments, analyzed and interpretation data and drafted the work. All authors read and approved the final manuscript. Acknowledgement We thank Dr. Nicholas Peraino (Wayne State University Lumigen Instrument Center) for mass spectrometry analysis and Dr. Charles P. Friedman for helpful discussions. This study received no funding. Data Availability All data generated or analyzed during the current study are included in this published article or in the Supplemental Material. References Polymeropoulos, M.H., et al. Mutation in the α-synuclein gene identified in families with Parkinson's disease. Science 276 , 2045–2047 (1997). Calabresi, P., et al. Alpha-synuclein in Parkinson’s disease and other synucleinopathies: From overt neurodegeneration back to early synaptic dysfunction. Cell Death Dis. 14 , 176 (2023). Narendra, D.P. & Youle, R.J. The role of PINK1–Parkin in mitochondrial quality control. Nature Cell Biol. 26 , 1639–1651 (2024). Ge, P., Dawson, V.L. & Dawson, T.M. PINK1 and Parkin mitochondrial quality control: a source of regional vulnerability in Parkinson’s disease. Molecular Neurodegeneration 15 , 20 (2020). Betarbet, R., et al. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nature Neurosci. 3 , 1301–1306 (2000). Costello, S., Cockburn, M., Bronstein, J., Zhang, X. & Ritz, B. Parkinson's disease and residential exposure to Maneb and Paraquat from agricultural applications in the Central Valley of California. Am. J. Epidemiol. 169 , 919–926 (2009). Paul, K.C., Cockburn, M., Gong, Y., Bronstein, J. & Ritz, B. Agricultural paraquat dichloride use and Parkinson’s disease in California’s Central Valley. Int. J. Epidemiol. 53 , dyae004 (2024). Wong, Y., et al. Neuronal vulnerability in Parkinson disease and putative therapeutics: Should the focus be on axonal and synaptic terminals? Movement Disorders 34 , 1406–1422 (2019). Pacelli, C., et al. Elevated mitochondrial bioenergetics and axonal arborization size are key contributors to the vulnerability of dopamine neurons. Current Biol. 25 , 2349–2360 (2015). Wimalasena, K. The inherent high vulnerability of dopaminergic neurons toward mitochondrial toxins may contribute to the etiology of Parkinson’s disease. Neural Regeneration Research 11 , 246–247 (2016). Surmeier, D.J., Obeso, J.A. & Halliday, G.M. Selective neuronal vulnerability in Parkinson disease. Nature Reviews Neuroscience 18 , 101–113 (2017). Masoud, S.T., et al. Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and L-DOPA reversible motor deficits. Neurobiology of Disease 74 , 66–75 (2015). Bucher, M.L., et al. Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson’s disease. NPJ Parkinsons Dis ease 6 , 34 (2020). Burbulla, L.F., et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's disease. Science 357 , 1255–1261 (2017). Herrera, A., Muñoz, P., Steinbusch, H.W.M. & Segura-Aguilar, J. Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s Disease? ACS Chem. Neurosci. 8 , 702 − 711 (2017). Biosa, A., et al. Dopamine oxidation products as mitochondrial endotoxins, a potential molecular mechanism for preferential neurodegeneration in Parkinson's Disease. ACS Chem. Neurosci. 9 , 2849 − 2858 (2018). Vauzour, D., Pinto, J.T., Cooper, A.J.L. & Spencer, J.P.E. The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signaling. Biochem. J. 463 , 41–52 (2014). Mehta, N.J., et al. Hypochlorite converts cysteinyl-dopamine into a cytotoxic product: A possible factor in Parkinson's disease. Free Radic. Biol. Med. 101 , 44–52 (2016). Zhang, F. & Dryhurst, G. Effects of L-cysteine on the oxidation chemistry of dopamine: New reaction pathways of potential relevance to idiopathic Parkinson's disease. J. Med. Chem. 37 , 1084–1098 (1994). Shen, X.M. & Dryhurst, G. Further insights into the influence of L-cysteine on the oxidation chemistry of dopamine: Reaction pathways of potential relevance to Parkinson’s disease. Chem. Res. Toxicol. 9 , 751–763 (1996). Li, H. & Dryhurst, G. Irreversible inhibition of mitochondrial complex I by 7- (2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): A putative nigral endotoxin of relevance to Parkinson’s disease. J. Neurochem. 69 , 1530–1541 (1997). Li, H., Shen, X.M. & Dryhurst, G. Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: Potential relevance to the pathogenesis of Parkinson’s disease. J. Neurochem. 71 , 2049–2062 (1998). Shen, X.M. & Dryhurst, G. Iron- and manganese-catalyzed autoxidation of dopamine in the presence of l-cysteine: Possible insights into iron- and manganese-mediated dopaminergic neurotoxicity. Chem. Res. Toxicol. 11 , 824–837 (1998). Mosca, L., Tempera, I., Lendaro, E., Di Francesco, L. & d’Erme, M. Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells. J. Neurosci. Res. 86 , 954–960 (2008). Wakamatsu, K., Murase, T., Zucca, F.A., Zecca, L. & Ito, S. Biosynthetic pathway to neuromelanin and its aging process. Pigment Cell & Melanoma Research 25 , 792–803 (2012). Marwah, P.K. Hypochlorite-mediated oxidation of dopamine: Implications in Parkinson’s disease and manganism. Ph.D. dissertation, Wayne State University (2021). Napolitano, A., Di Donato, P. & Prota, G. New regulatory mechanisms in the biosynthesis of pheomelanins: Rearrangement vs. redox exchange reaction routes of a transient 2H-1,4-benzothiazine-o-quinonimine intermediate. Biochim. Biophys. Acta 1475 , 47–54 (2000). Napolitano, A., Memoli, S. & Prota, G. A new insight in the biosynthesis of pheomelanins: Characterization of a labile 1,4-benzothiazine intermediate. J. Org. Chem. 64 , 3009–3011 (1999). Henrich, M.T., Oertel, W.H., Surmeier, D.J. & Geibl, F.F. Mitochondrial dysfunction in Parkinson’s disease – a key disease hallmark with therapeutic potential. Molecular Neurodegeneration 18 , 83 (2023). Adetuyi, O.A. & Wimalasena, K. Exposure to Mitochondrial Toxins: An in vitro study of energy depletion and oxidative stress in driving dopaminergic neuronal death in MN9D cells. Toxics 13 , 637 (2025). Nagatsu, T., et al. The role of tyrosine hydroxylase as a key player in neuromelanin synthesis and the association of neuromelanin with Parkinson’s disease. J. Neural Transmission 130 , 611–625 (2023). Rosengren, E., Linder-Eliasson, E. & Carlsson, A. Detection of 5-S-cysteinyldopamine in human brain. J. Neural Transmission 63 , 247–253 (1985). Fornstedt, B., Rosengren, E. & Carlsson, A. Occurrence and distribution of 5-S-cysteinyldopamine, dopa and dopac in the brains of eight mammalian species. Neuropharmacol. 25 , 451–454 (1986). Fornstedt, B., Bergh, I., Rosengren, E. & Carlsson, A. An improved HPLC-electrochemical detection method for measuring brain levels of 5-S-cysteinyldopamine, 5-S-cysteinyl-3,4-dihydroxyphenylalanine, and 5-S-cysteinyl-3,4-dihydroxyphenylacetic acid. J. Neurochem. 54 , 578–586 (1990). Malagelada, C. & Greene, L.A. PC12 Cells as a model for parkinson's disease research. In: Parkinson’s disease: Molecular and therapeutic insights from model systems. Edited by R. Nass and S. Przedborski. Cambridge, MA: Academic; p. 375–387 (2008). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9280779","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624331648,"identity":"16a4f3b4-2388-4a5c-bd0b-5e4839209079","order_by":0,"name":"Praneet Kaur Marwah","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Praneet","middleName":"Kaur","lastName":"Marwah","suffix":""},{"id":624331650,"identity":"55d53e5b-651f-4aae-8202-b5a1ec86828d","order_by":1,"name":"Lin Rayes","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Rayes","suffix":""},{"id":624331658,"identity":"43043f1b-3fc3-4818-8fa2-3adc08878cea","order_by":2,"name":"Sanjana Aiella","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Sanjana","middleName":"","lastName":"Aiella","suffix":""},{"id":624331659,"identity":"e89a1c66-df22-435e-b519-e4cf28786f1e","order_by":3,"name":"Alexa Atty","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Alexa","middleName":"","lastName":"Atty","suffix":""},{"id":624331661,"identity":"28714954-011c-4701-8e37-d9183663a851","order_by":4,"name":"Ali Rashid","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Rashid","suffix":""},{"id":624331663,"identity":"b6264dce-30d6-470c-b116-49f5cd31b4c9","order_by":5,"name":"Karandeep Singh","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Karandeep","middleName":"","lastName":"Singh","suffix":""},{"id":624331664,"identity":"8f92a2f0-16c3-4e69-bd9c-819210262021","order_by":6,"name":"Eduardo Palomino","email":"","orcid":"","institution":"Wayne State University","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Palomino","suffix":""},{"id":624331668,"identity":"132604a1-b963-40cb-839c-d8dc7ec305c0","order_by":7,"name":"David Njus","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDCCAwwJDAwVMB4b0VrOkKiFgYGxjRQtfLcbnkn+nHdY3uB28wGGD2WHCWuRvHMgTZp322HDDXeOJTDOOEeEFoMbCWnSjNtuM264kWPAzNtGpBbJn3Nu22+4kf+B+S+xWiR4G24nAm1hYGYkRovkjYRka55j/5Nn3kgzONhzLp2wFr4bOYk3f9Sk2fbdSH744EeZNWEtDAw8CWBK4QAkjogB7BCF8g1Eqh8Fo2AUjIKRBwDuPUSiIZWqSAAAAABJRU5ErkJggg==","orcid":"","institution":"Wayne State University","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Njus","suffix":""}],"badges":[],"createdAt":"2026-03-31 13:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9280779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9280779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107189055,"identity":"0c2ba62f-dcaa-46df-921c-e9bdda06a863","added_by":"auto","created_at":"2026-04-17 20:16:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65307,"visible":true,"origin":"","legend":"\u003cp\u003eBiosynthesis of neuromelanin intermediates.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/f0639d517d78b7d1b8f2afb2.png"},{"id":107482760,"identity":"1b490bf0-89c1-4c0a-b2f4-daeea79a923a","added_by":"auto","created_at":"2026-04-22 02:24:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35892,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of DHBT-1 is less than expected but enhanced by BT-2. A) Purified DHBT-1 is cytotoxic but less than its precursor cysteinyl-dopamine and unpurified hypochlorite-oxidized cysteinyl-dopamine. DHBT-1, cysteinyl-dopamine (CysDA), and cysteinyl-dopamine treated with equimolar NaOCl (HOCD) were added separately at the indicated concentrations to PC12 cells. The percentage of live cells was counted after 48 hours. B) BT-2 potentiates the toxicity of DHBT-1. The indicated concentration of DHBT-1 was added to PC12 cells with or without 100 µM BT-2. The percentage of live cells was counted after 48 hours. All points are the average (±SD) of three replicate samples.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/600ff3401726f9d8fd2dc4dd.png"},{"id":107189056,"identity":"a8fbea56-340a-4e54-ad95-ecf1e0bc6892","added_by":"auto","created_at":"2026-04-17 20:16:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46073,"visible":true,"origin":"","legend":"\u003cp\u003eBT-2 potentiates the toxicity of sublethal concentrations of the mitochondrial complex I inhibitors rotenone and Paraquat but not of complex III or complex V inhibitors (antimycin A or oligomycin respectively). The indicated concentration of each mitochondrial inhibitor was added to PC12 cells with or without BT-2. A) Rotenone (±80 µM BT-2), B) Paraquat (±108 µM BT-2), C) antimycin A (±100 µM BT-2) and D) oligomycin (±100 µM BT-2). The percentage of live cells was counted after 48 hours. All points are the average (±SD) of three replicate samples.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/45dd937e4536a588c602a0e7.png"},{"id":107483372,"identity":"ca2a4c20-31ef-4b2b-83f5-9816127a7e0b","added_by":"auto","created_at":"2026-04-22 02:27:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62642,"visible":true,"origin":"","legend":"\u003cp\u003eStability of BT-2 and Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; A)\u0026nbsp; Dimerization of BT-2 produces the violet-colored decarboxy trichochrome F.\u0026nbsp; Me\u003csub\u003e2\u003c/sub\u003eBT-2 is stabilized by the fact that it cannot undergo the oxidation step marked by the red X. B) Normalized absorption spectra of DHBT-1, BT-2 and Me\u003csub\u003e2\u003c/sub\u003eBT-2 in H\u003csub\u003e2\u003c/sub\u003eO. C) Absorption spectra of 0.6 mM BT-2 after incubation in 0.5 M HCl for 0, 10, 20, 30, 60 and 120 min.\u0026nbsp; D) Formation of trichochrome from BT-2, DHBT-1 and Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; Each compound (0.6 mM final concentration) was added to 0.5 M HCl at t=0, and absorbance at 580 nm was recorded.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/22628859bd94371282ea5f6e.png"},{"id":107189059,"identity":"1754b6d5-860f-4702-b6d9-593839efc0b6","added_by":"auto","created_at":"2026-04-17 20:16:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50926,"visible":true,"origin":"","legend":"\u003cp\u003eMe\u003csub\u003e2\u003c/sub\u003eBT-2 potentiates the cytotoxicity of rotenone, but DHBT-1 does not. A)\u0026nbsp; The indicated concentration of DHBT-1 was added to PC12 cells with or without 100 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; B) The indicated concentration of rotenone was added to PC12 cells with or without 134 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; C) The indicated concentration of rotenone was added to PC12 cells with or without 0.5 mM DHBT-1.\u0026nbsp; In all cases, the percentage of live cells was counted after 48 hours.\u0026nbsp; All points are the average (±SD) of three replicate samples.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/bbceac4c60c1c8596d7ce7c1.png"},{"id":107483655,"identity":"087346a2-3a61-418a-bd25-cf9fe946279b","added_by":"auto","created_at":"2026-04-22 02:28:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":54748,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Me\u003csub\u003e2\u003c/sub\u003eBT-2 on toxicity of mitochondrial inhibitors.\u0026nbsp; A-C)\u0026nbsp; The indicated concentration of each inhibitor was added to PC12 cells with or without Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; A) Paraquat (±137 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2), B) antimycin A (±137 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2) and C) oligomycin (±100 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2). In all cases, the percentage of live cells was counted after 48 hours.\u0026nbsp; All points are the average (±SD) of three replicate samples.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/7e4b9381d53a1fa48ee87219.png"},{"id":107189060,"identity":"3b12c893-9d6a-44e5-a567-237db2f6fd36","added_by":"auto","created_at":"2026-04-17 20:16:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":53907,"visible":true,"origin":"","legend":"\u003cp\u003eConditions affecting the toxicity of DHBT-1 and cotoxicity of Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; A)\u0026nbsp; DHBT-1 at the indicated concentrations was added to PC12 cells in DMEM/F-12 medium or Ham’s F-12K (Kaighn’s) medium.\u0026nbsp; The percentage of live cells was counted after 48 hours.\u0026nbsp; B) Me\u003csub\u003e2\u003c/sub\u003eBT-2 at the indicated concentrations was added to PC12 cells in DMEM/F-12 medium or Ham’s F-12K (Kaighn’s) medium.\u0026nbsp; The percentage of live cells was counted after 48 hours.\u0026nbsp; C) Paraquat (0 or 1.16 mM) was added to PC12 cells in DMEM/F-12 medium or Ham’s F-12K (Kaighn’s) medium in the presence or absence of 77 µM Me\u003csub\u003e2\u003c/sub\u003eBT-2.\u0026nbsp; The percentage of live cells was counted after 48 hours. All points are the average (±SD) of two or three replicate samples.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/620ee4d6dfef84448c8fd48b.png"},{"id":109067276,"identity":"86751912-bafb-44a1-b9ff-4b61a20b2b41","added_by":"auto","created_at":"2026-05-12 09:30:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":886936,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/12526bc8-dc87-4b18-8318-51a7ad4b10e4.pdf"},{"id":107189054,"identity":"4f3bd7a1-61f0-46b1-ab5e-8c716adc99b9","added_by":"auto","created_at":"2026-04-17 20:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":231400,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9280779/v1/48229a2e166f66b15cacb3b5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dopamine-Derived Cotoxins and the Unique Vulnerability of Dopaminergic Neurons in Parkinson’s Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe movement disorders characteristic of Parkinson’s disease result from the death of dopaminergic neurons in the substantia nigra. \u0026nbsp;Many factors contributing to the disease or its animal models have been identified. \u0026nbsp; These include mutations in proteins such as α-synuclein, Parkin and PINK1\u003csup\u003e1-4\u003c/sup\u003e and mitochondrial inhibitors such as rotenone and Paraquat.\u003csup\u003e5-7\u003c/sup\u003e The mutant proteins are expressed not just in dopaminergic neurons, however, and the mitochondrial inhibitors should affect nearly all cells in the body. Although Parkinson’s disease has been studied intensely, a central question remains: Why are dopaminergic neurons uniquely vulnerable to these stresses?\u003csup\u003e\u0026nbsp;8-11\u003c/sup\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The extensive arborization of dopaminergic neurons and the attendant stress of dispersed bioenergetic and synaptic functions has been suggested.\u003csup\u003e8,9\u003c/sup\u003e\u0026nbsp; There is also considerable evidence, however, that dopamine degradation products play a crucial role.\u003csup\u003e12-16\u003c/sup\u003e\u0026nbsp; Transgenic mice overexpressing the dopamine transporter in dopamine neurons specifically display a loss of midbrain dopamine neurons and motor deficits reversed by L-DOPA. \u0026nbsp;This is associated with an increase in the dopamine oxidation product, cysteinyl-dopamine.\u003csup\u003e12\u003c/sup\u003e Knockdown of the vesicular monoamine transporter (VMAT2) in the substantia nigra of rats causes an increase in cytosolic dopamine followed by degeneration of nigrostriatal dopaminergic neurons, deficits in dopamine-mediated behaviors, and the formation of aberrant α-synuclein.\u003csup\u003e13\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the cellular level, treating SH-SY5Y cells with dopamine oxidized using tyrosinase results in mitochondrial membrane depolarization, reduction of ATP synthesis, opening of the mitochondrial transition pore and ultimately cell death.\u003csup\u003e16\u003c/sup\u003e Primary cultures of mouse cortical neurons are killed by cysteinyl-dopamine and/or its breakdown products.\u003csup\u003e17\u003c/sup\u003e PC-12 cells are killed by a cytotoxic preparation obtained when cysteinyl-dopamine is treated with sodium hypochlorite.\u003csup\u003e18\u003c/sup\u003e Scavenging hypochlorous acid protects against the cytotoxicity of cysteinyl-dopamine but not against hypochlorous acid-treated cysteinyl-dopamine (HOCD), suggesting that the cytoxic activity is downstream of cysteinyl-dopamine.\u003c/p\u003e\n\u003cp\u003eDespite this evidence, there have been relatively few studies attempting to identify specific dopamine-derived toxins and their mechanism of action. Dryhurst’s group identified 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2\u003cem\u003eH\u003c/em\u003e-benzo[b][1,4]thiazine-3-carboxylic acid or DHBT-1 as the major oxidation product of cysteinyl-dopamine.\u003csup\u003e19-21\u003c/sup\u003e They also showed that it is an inhibitor of complex I of the mitochondrial respiratory chain but did not assess cytotoxicity.\u003csup\u003e21,22\u003c/sup\u003e The same group identified 7-(2-aminoethyl)-\u003cem\u003e2H\u003c/em\u003e-benzo[b][1,4]thiazin-5-ol\u0026nbsp;or\u0026nbsp;BT-2 but found it too unstable to pursue.\u003csup\u003e22,23\u003c/sup\u003e In fact, significant obstacles to studying these compounds may be that they are unstable and tend to polymerize and that current methodology for preparing them is laborious and requires specialized chemical facilities. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we present rapid and simple procedures for preparing and purifying DHBT-1 and BT-2. We also describe the synthesis of 2,2-dimethyl-BT-2, a very stable analogue of BT-2. Using these compounds, we test the hypothesis that they increase the toxicity of mitochondrial complex I inhibitors and account for the unique vulnerability of dopaminergic neurons to stresses implicated in Parkinson’s disease. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCysteinyl-dopamine is a primary oxidation product of dopamine \u003cem\u003ein vivo\u003c/em\u003e and a precursor of neuromelanin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Previously, we showed that a cytotoxic preparation is obtained when dopamine is oxidized by tyrosinase in the presence of cysteine, and the resulting cysteinyl-dopamine is then treated with sodium hypochlorite.\u003csup\u003e18\u003c/sup\u003e To identify and characterize the active compounds in this preparation, we scaled up the synthesis of cysteinyl-dopamine by oxidizing dopamine using ceric ammonium nitrate instead of tyrosinase.\u003csup\u003e24\u003c/sup\u003e This preparation of cysteinyl-dopamine confirms that cysteinyl-dopamine and hypochlorous acid-oxidized cysteinyl-dopamine (HOCD) are cytotoxic to PC12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon purification, the major component of HOCD was determined to be DHBT-1, a dihydrobenzothiazine previously recognized by others as a major product of cysteinyl-dopamine oxidized by different methods.\u003csup\u003e19,25\u003c/sup\u003e DHBT-1, identified by its absorption spectrum and confirmed by high-resolution mass spectrometry, is obtained from cysteinyl-dopamine with very high yield. DHBT-1 kills PC12 cells; however, it is much less cytotoxic than either cysteinyl-dopamine or HOCD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe loss of toxicity upon purification of DHBT-1 suggested the existence of a cotoxin augmenting DHBT-1 toxicity. In fact, when DHBT-1 is purified by reverse-phase chromatography, a cotoxin activity elutes after DHBT-1.\u003csup\u003e26\u003c/sup\u003e High resolution mass spectrometry identified the chemical formula of the cotoxin as C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eOS consistent with BT-2. Although BT-2 can be prepared by oxidizing cysteinyl-dopamine with sodium hypochlorite, Napolitano et al. reported an efficient synthesis of the cysteinyl-L-DOPA analogue using potassium ferricyanide as the oxidizing agent at 37\u0026deg;C. \u003csup\u003e27\u003c/sup\u003e Using this procedure, we prepared BT-2 at a low concentration (~\u0026thinsp;2 mM) to minimize polymerization of the product. This preparation of BT-2 is not very toxic itself, but it greatly increases the toxicity of DHBT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe fact that BT-2 enhances the lethality of DHBT-1 raises the possibility that it might enhance the toxicity of other factors implicated in Parkinson\u0026rsquo;s disease, thereby contributing to the unique vulnerability of dopaminergic neurons to these factors. Because DHBT-1 has been reported to inhibit complex I of the mitochondrial respiratory chain,\u003csup\u003e21,22\u003c/sup\u003e we tested BT-2 to see if it potentiates the toxicity of other complex I inhibitors. Indeed, rotenone and Paraquat are quite toxic in the presence of BT-2 even at concentrations at which they otherwise have negligible effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). To see whether this cotoxicity is unique to complex I inhibitors or applies to any stress, we also tested the effect of BT-2 on the toxicity of the complex III inhibitor antimycin A and the ATP synthase inhibitor oligomycin. BT-2 increases the toxicity of these compounds, but only at concentrations that already cause significant cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs reported by others,\u003csup\u003e19,28\u003c/sup\u003e BT-2 is not very stable. To analyze its biological properties, we synthesized, purified and tested the cytotoxicity of the compound on the same day. One reason for the instability of BT-2 is its tendency to dimerize at the C2 position especially at acidic pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). While BT-2 itself absorbs in the ultraviolet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), it decomposes to a purple product with an absorbance maximum at 580 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) consistent with formation of decarboxylated trichochrome F.\u003csup\u003e23,27\u003c/sup\u003e In 0.5 M HCl, BT-2 converts fairly rapidly to this trichochrome, but DHBT-1 does not (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA stable analogue of BT-2 may be synthesized by using using penicillamine instead of cysteine. This adds two methyl groups at the C2 position of BT-2 yielding 7-(2-aminoethyl)- 2,2-dimethyl-\u003cem\u003e2H\u003c/em\u003e-benzo[b][1,4]thiazin-5-ol (Me\u003csub\u003e2\u003c/sub\u003eBT-2). The absorption spectrum of Me\u003csub\u003e2\u003c/sub\u003eBT-2 is very similar to that of BT-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), and both have a higher absorbance at 200 nm than DHBT-1. A significant difference is that Me\u003csub\u003e2\u003c/sub\u003eBT-2 does not convert to the trichochrome in 0.5 M HCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In fact, Me\u003csub\u003e2\u003c/sub\u003eBT-2 is quite stable and can be kept in aqueous solution for several weeks at 4\u0026deg;C. Moreover, because Me\u003csub\u003e2\u003c/sub\u003eBT-2 is stable under acidic conditions, it can be synthesized in high yield in one step by oxidizing dopamine in the presence of penicillamine in HCl.\u003c/p\u003e \u003cp\u003eLike BT-2, Me\u003csub\u003e2\u003c/sub\u003eBT-2 potentiates the toxicity of DHBT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and rotenone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Given that BT-2 and Me\u003csub\u003e2\u003c/sub\u003eBT-2 both potentiate the toxicity of rotenone, one might ask whether DHBT-1 also exhibits this cotoxin activity. DHBT-1 does not increase the toxicity of rotenone, however, (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), so DHBT-1 is a toxin but not a cotoxin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen tested with other mitochondrial inhibitors, Me\u003csub\u003e2\u003c/sub\u003eBT-2 shows the same pattern of cotoxicity as BT-2. Like BT-2, it amplifies the toxicities of rotenone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and Paraquat (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), but it has less effect on the toxicity of antimycin A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and no effect with oligomycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the toxicity of DHBT-1 depends on the medium. It is toxic at a significantly lower concentration when PC12 cells are treated in DMEM/F12 medium than when they are in Ham\u0026rsquo;s F12K medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Because Me\u003csub\u003e2\u003c/sub\u003eBT-2 is stable at higher concentrations than BT-2, its toxicity can be measured. It is actually toxic at somewhat lower concentrations than DHBT-1, but its toxicity does not depend on the medium in the same way (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Furthermore, the cotoxin activity of Me\u003csub\u003e2\u003c/sub\u003eBT-2 is more evident when tested in Ham\u0026rsquo;s F12K medium than in DMEM/F12 medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The same seems to be true of BT-2. Therefore, cotoxicity of Me\u003csub\u003e2\u003c/sub\u003eBT-2 and BT-2 and toxicity of DHBT-1 are both affected by the composition of the medium, but in different ways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHallmark characteristics of Parkinson\u0026rsquo;s disease include the loss of dopaminergic neurons in the substantia nigra, motor deficits caused by the impairment of this motor circuit, and the appearance of α-synuclein deposits in Lewy bodies. A number of reports suggest that dopamine oxidation and mitochondrial dysfunction play integral roles in this neurodegeneration.\u003csup\u003e5,12\u0026ndash;14,29,30\u003c/sup\u003e As mentioned, cytoplasmic oxidation of dopamine may be increased by overexpressing the cell membrane dopamine transporter or by knocking down the vesicular monoamine transporter. Both of these lead to loss of dopaminergic neurons, development of motor deficits and an increase in α-synuclein.\u003csup\u003e12,13\u003c/sup\u003e Similarly, disruption of mitochondrial respiration by chronic treatment with rotenone creates a rat model of Parkinson\u0026rsquo;s disease in which Lewy bodies are formed, movement is impaired, and dopaminergic neurons are lost.\u003csup\u003e5\u003c/sup\u003e Mitochondrial dysfunction caused by a DJ-1 loss-of-function mutation leads to dopamine oxidation and α-synuclein accumulation.\u003csup\u003e14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe identity of the dopamine oxidation products mediating this degeneration is still a matter of debate. The ultimate product of dopamine oxidation is neuromelanin, the black pigment that gives the substantia nigra its name. Neuromelanin is an amorphous polymer incorporating products of dopamine and cysteinyl-dopamine. Its biosynthesis parallels that of pheomelanin except that the latter is made from L-DOPA and cysteine. The initial steps in the biosynthesis of neuromelanin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are well known, and the first intermediates are cysteinyl-dopamine, DHBT-1 and BT-2.\u003csup\u003e20,25,31\u003c/sup\u003e The biological effects of these intermediates have not received much attention, however, a significant obstacle being that they naturally polymerize especially at high concentrations. For this reason, it is difficult to synthesize, purify and store these compounds, especially BT-2, in a form suitable for cytoxicity studies.\u003c/p\u003e \u003cp\u003eDopamine oxidizes slowly but spontaneously in the presence of O\u003csub\u003e2\u003c/sub\u003e to form the dopamine quinone. The dopamine quinone may cyclize and oxidize to form aminochrome, or it may react with thiols such as cysteine to form cysteinyl-dopamine. The reaction with cysteine is quite fast, and the product, cysteinyl-dopamine, is relatively stable. In contrast, aminochrome decomposes rather quickly. Consistent with this, cysteinyl-dopamine is found in the brain\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e but the detection of aminochrome has not been reported.\u003c/p\u003e \u003cp\u003eAlthough cysteinyl-dopamine kills cells,\u003csup\u003e17\u003c/sup\u003e we have argued that this toxicity is attributable to its conversion to other products by endogenous oxidants such as hypochlorous acid.\u003csup\u003e18\u003c/sup\u003e Taurine, which scavenges hypochlorous acid, protects against the cytotoxicity of cysteinyl-dopamine but not against HOCD, cysteinyl-dopamine already oxidized with hypochlorous acid.\u003c/p\u003e \u003cp\u003eThe primary products of cysteinyl-dopamine oxidation are DHBT-1 and BT-2. These are alternative products: less oxidation favors DHBT-1; more oxidation yields BT-2. Studying melanin biosynthesis, Napolitano et al. demonstrated this dichotomy using L-DOPA instead of dopamine with ferricyanide as the oxidizer.\u003csup\u003e27\u003c/sup\u003e To make DHBT-1, an oxidizer is required only to initiate oxidation of cysteinyl-dopamine, thereby forming a catalytic amount of o-quinone imine. The reduction potentials are such that the o-quinone imine will then oxidize cysteinyl-dopamine and itself be reduced to DHBT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This cycling results in complete conversion of cysteinyl-dopamine to DHBT-1. More oxidizer actually lowers the yield of DHBT-1 because it oxidizes more cysteinyl-dopamine making it unavailable to reduce the o-quinone imine to DHBT-1.\u003c/p\u003e \u003cp\u003eHigher concentrations of oxidizer increase the formation of BT-2, because greater oxidation of cysteinyl-dopamine forces the o-quinone imine to to take the less favored pathway leading to BT-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). BT-2 oxidizes readily, so it is unstable and decomposes over a period of hours. It may dimerize to form a trichochrome or react with other thiols like cysteine or glutathione. Our synthesis of BT-2 uses a lower concentration of cysteinyl-dopamine, because the lower concentration of product slows dimerization and improves yield and stability of BT-2. It also limits the amount that may be synthesized, however, and the concentration of stock solutions.\u003c/p\u003e \u003cp\u003eThe oxidation of BT-2 may be prevented by adding methyl groups to the C2 carbon. Napolitano et al. took advantage of this approach, using D-penicillamine instead of cysteine to produce more a stable product with L-DOPA.\u003csup\u003e28\u003c/sup\u003e They were interested in characterizing intermediates in the formation of melanin, however, and not in their biological activities. Interestingly, the dimethylated analogue of BT-2 (Me\u003csub\u003e2\u003c/sub\u003eBT-2) can be synthesized from penicillamine and dopamine in one step using a slightly higher concentration of oxidant (ceric ammonium nitrate) and lower concentration of thiol (penicillamine). Dopamine oxidation then leads to formation of penicillaminyl-dopamine, and the latter is further oxidized in HCl to Me\u003csub\u003e2\u003c/sub\u003eBT-2. Because Me\u003csub\u003e2\u003c/sub\u003eBT-2 cannot dimerize to a trichochrome, the reaction stops there. BT-2 cannot be made in the same way, because it goes on to dimerize and form other products.\u003c/p\u003e \u003cp\u003eHaving the capacity to make DHBT-1, BT-2 and the cotoxin Me\u003csub\u003e2\u003c/sub\u003eBT-2, it is now possible to test their involvement in Parkinson\u0026rsquo;s disease. Are these toxins present in dopaminergic neurons at concentrations physiologically relevant to Parkinson\u0026rsquo;s disease? We found that DHBT-1 is toxic at millimolar concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while BT-2 acts as a cotoxin at 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). While these concentrations may not seem especially potent, several issues should be considered. First, most people do not get Parkinson\u0026rsquo;s disease, suggesting that these compounds normally polymerize to neuromelanin and do not reach toxic concentrations. Second, we observed toxicity in a majority of cells after two days of treatment. Parkinson\u0026rsquo;s disease kills dopaminergic neurons over decades, so these compounds may cause damage that accumulates over time. Alternatively, toxicity may occur episodically with these toxins reaching higher concentrations transiently and in specific locations. In addition, the toxic dose seems to depend on conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), so there may be factors that render some individuals more susceptible to these toxins. Finally, the cotoxin is synergistic and is only needed to augment the toxicity of other stresses.\u003c/p\u003e \u003cp\u003eTherefore, important questions are: 1) Are there conditions or events that increase the concentrations or toxicities of DHBT-1 and BT-2 in some people? 2) How do these compounds synergize with other factors linked to Parkinson\u0026rsquo;s disease to cause neurotoxicity? 3) How does BT-2 cause cotoxicity and might this identify potential targets for therapeutic intervention?\u003c/p\u003e \u003cp\u003eIdiopathic cases of Parkinson\u0026rsquo;s disease may involve a diminished ability to clear DHBT-1 and BT-2 or an increased sensitivity to their toxicity. Understanding this connection could be significant both as a diagnostic indicator of susceptibility to Parkinson\u0026rsquo;s disease and for the identification of potential therapeutic targets. While we do not yet know how conditions affect the concentrations of these toxins or their interactions with their targets, this clearly requires investigation.\u003c/p\u003e \u003cp\u003eA significant unanswered question is why are dopaminergic neurons exceptionally vulnerable to other factors linked to Parkinson\u0026rsquo;s disease including aggregates of α-synuclein and mutations in proteins such as Parkin, PINK1 and LRRK2. If the cotoxin BT-2 potentiates the toxicities of these factors, then it could be the missing piece that answers this question. The availability of the stable BT-2 analogue, Me\u003csub\u003e2\u003c/sub\u003eBT-2, now makes if more practical to test the effect of the cotoxin on the toxicities of these other factors.\u003c/p\u003e \u003cp\u003eFinally, how does BT-2 cause cotoxicity? Knowing how the cotoxin acts will not only elucidate the mechanism of Parkinson\u0026rsquo;s disease, but it may identify potential therapeutic interventions. To date, cell culture has not provided much information about the molecular processes leading to the death of dopaminergic neurons, perhaps because studies have not included the dopamine-derived toxins DHBT-1 and BT-2 but have relied instead on artificial dopaminergic toxins like 6-hydroxydopamine or 1-methyl-4-phenylpyridinium (MPP+). The availability of Me\u003csub\u003e2\u003c/sub\u003eBT-2 will make it more practical for researchers to use cell culture rather than animals to investigate the interaction of cotoxin with chemicals and mutations implicated in Parkinson\u0026rsquo;s disease. This in turn may provide an efficient, high-throughput, cell-culture screen for treatments that block the toxicity of these Parkinsons\u0026rsquo;s agents and thereby have the potential to slow or stop the progression of the disease.\u003c/p\u003e \u003cp\u003eIn conclusion, we have shown that BT-2 is a cotoxin that potentiates the toxicities of mitochondrial inhibitors including rotenone, Paraquat and the endogenous toxin DHBT-1. Thus, BT-2 and DHBT-1 may be missing pieces in the puzzle of Parkinson\u0026rsquo;s disease, and knowing how they fit into the processes leading to the death of dopaminergic neurons may lead to a more complete understanding of the progression of the disease and better-informed design of interventions to slow or stop it.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSodium hypochlorite, ceric ammonium nitrate, dopamine HCl, L-cysteine, and D-penicillamine were purchased from Sigma/Aldrich, as were rotenone, antimycin A, methyl viologen (Paraquat) and oligomycin.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eChemical Syntheses\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eCysteinyl dopamine\u003c/em\u003e: Ten ml of ceric ammonium nitrate (0.4 M in 0.25 N HCl) was added to 5 ml of dopamine (0.4 M in 0.25 N HCl). This was immediately added to 10 ml of L-cysteine (0.24 M in 0.25 N HCl), and the mixture was applied to a Sep-Pak 35 cc C18 Vac cartridge and eluted with 52 ml of 0.1 M HCl. Cysteinyl dopamine was identified and quantified by its UV absorption spectrum (extinction coefficients: 3,810 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 254 nm and 2,860 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 293 nm).\u003csup\u003e24\u003c/sup\u003e Yield averaged about 45%.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDHBT-1\u003c/em\u003e: Cysteinyl-dopamine (0.4 mmol) was mixed with 35 ml of 50 mM potassium phosphate, pH 6.8. Six percent NaOCl (0.5 ml or 0.4 mmol) was added. The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 26 ml of H\u003csub\u003e2\u003c/sub\u003eO followed by 52 ml of 10% ethanol. DHBT-1 was identified and quantified by its UV absorption spectrum (extinction coefficients: 11,480 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 234 nm and 1,905 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 307 nm).\u003csup\u003e19\u003c/sup\u003e Yield was close to 100%. Identification was confirmed by high resolution mass spectrometry (Supplementary Fig.\u0026nbsp;1) which gave M\u0026thinsp;+\u0026thinsp;z\u0026thinsp;=\u0026thinsp;255.0795 (theoretical\u0026thinsp;=\u0026thinsp;255.0803) consistent with a chemical formula of C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMe\u003csub\u003e2\u003c/sub\u003eBT-2\u003c/strong\u003e \u003cp\u003eTen ml of ceric ammonium nitrate (0.42 M in 1 N HCl) was added to 5 ml of dopamine (0.4 M in 1 N HCl). This was immediately added to 10 ml of D-penicillamine (0.2 M in 1 N HCl). The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 39 ml of 4% ethanol followed by 72 ml of 10% ethanol. The UV absorption spectrum in H\u003csub\u003e2\u003c/sub\u003eO has maxima at 232 nm (ϵ = 17,400 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 302 nm (ϵ = 2,060 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Yield was about 30%. Identification was confirmed by high resolution mass spectrometry (Supplementary Fig.\u0026nbsp;2) which gave M\u0026thinsp;+\u0026thinsp;z\u0026thinsp;=\u0026thinsp;237.1053 (theoretical\u0026thinsp;=\u0026thinsp;237.1062) consistent with a chemical formula of C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eOS).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBT-2\u003c/strong\u003e \u003cp\u003eCysteinyl-dopamine (0.08 mmol) was mixed with 40 ml of 50 mM potassium phosphate, pH 6.8 at 37\u0026deg;C. Then 3.6 ml of 100 mM potassium ferricyanide (0.36 mmol) was added at 37\u0026deg;C with stirring. The resulting solution was applied to a Sep-Pak 35 cc C18 Vac cartridge eluted with 26 ml of H\u003csub\u003e2\u003c/sub\u003eO followed by 52 ml of 21% ethanol. BT-2 was identified by its UV absorption spectrum (maxima at 225 nm and 300 nm). Because BT-2 is unstable on drying, its concentration was estimated using the same extinction coefficient as its analogue Me\u003csub\u003e2\u003c/sub\u003eBT-2 (ϵ = 17,400 M\u003csup\u003e\u0026minus;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 225 nm). Yield was about 7%. For toxicity experiments, BT-2 was added to cell cultures immediately following synthesis and purification.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eSpectrometry\u003c/h3\u003e\n\u003cp\u003eConcentrations of products were determined from UV absorption spectra recorded on a Molecular Devices SpectraMax M2 operated in cuvette mode. Spectra were recorded in H\u003csub\u003e2\u003c/sub\u003eO, and concentrations calculated using the extinction coefficients listed above. High resolution mass spectrometry was performed on a Thermo Scientific LTQ Orbitrap XL spectrometer in the Lumigen Instrument Center.\u003c/p\u003e\n\u003ch3\u003eCytotoxicity\u003c/h3\u003e\n\u003cp\u003ePC12 cells (ATCC Cat# CRL-1721.1, RRID: CVCL F659) were used to test cytotoxicity because they are an adrenergic cell line and are commonly used as a model for Parkinson\u0026rsquo;s disease studies.\u003csup\u003e35\u003c/sup\u003e Furthermore, we used them earlier to demonstrate the toxicity of hypochlorite-oxidized cysteinyl-dopamine and its synergy with rotenone.\u003csup\u003e18\u003c/sup\u003e PC12 cells (were cultured in 6-well plates in 2 ml of Ham\u0026rsquo;s F-12K (Kaighn\u0026rsquo;s) medium, supplemented with 15% heat-inactivated horse serum, 2.5% fetal bovine serum and 1% penicillin/streptomycin/ glutamine at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Toxins were added when cells reached confluence. After 48 hours of treatment, cell viability was measured using the trypan blue exclusion assay. Cells were detached using 0.1% trypsin, centrifuged, and resuspended in 0.2% trypan blue in Hanks Buffered Salt Solution. After 3 min, live and dead cells were counted using a Countess Automated Cell counter (Invitrogen).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor experiments testing cotoxicity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the statistical significance of the difference between slopes of lines with or without cotoxin was determined using GraphPad Prism. This calculates a two-tailed p value testing the null hypothesis that the slopes are identical. Significance is indicated as ns (p\u0026thinsp;\u0026ge;\u0026thinsp;0.05), *(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), **(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), ***(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ****(p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBT-2, 7-(2-aminoethyl)-\u003cem\u003e2H\u003c/em\u003e-benzo[b][1,4]thiazin-5-ol; DHBT-1, 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2\u003cem\u003eH\u003c/em\u003e-benzo[b][1,4]thiazine-3-carboxylic acid; Me\u003csub\u003e2\u003c/sub\u003eBT-2, 7-(2-aminoethyl)-2,2-dimethyl-\u003cem\u003e2H\u003c/em\u003e-benzo[b][1,4]thiazin-5-ol; CysDA, Cysteinyl-dopamine; HOCD, hypochlorite-oxidized cysteinyl-dopamine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.K.M designed experiments and acquired, analyzed, and interpreted data. L.R. prepared compounds and conducted toxicity experiments. S.A., A.A., A.R. and K.S. prepared compounds and performed experiments. E.P. contributed to development of chemical synthesis procedures and performed chemical analysis. D.N. designed and supervised chemical syntheses and cytotoxicity experiments, analyzed and interpretation data and drafted the work. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Nicholas Peraino (Wayne State University Lumigen Instrument Center) for mass spectrometry analysis and Dr. Charles P. Friedman for helpful discussions. This study received no funding.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during the current study are included in this published article or in the Supplemental Material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePolymeropoulos, M.H., et al. Mutation in the α-synuclein gene identified in families with Parkinson's disease. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e276\u003c/b\u003e, 2045–2047 (1997).\u003c/li\u003e\n\u003cli\u003eCalabresi, P., et al. Alpha-synuclein in Parkinson’s disease and other synucleinopathies: From overt neurodegeneration back to early synaptic dysfunction. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 176 (2023).\u003c/li\u003e\n\u003cli\u003eNarendra, D.P. \u0026amp; Youle, R.J. The role of PINK1–Parkin in mitochondrial quality control. \u003cem\u003eNature Cell Biol.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 1639–1651 (2024).\u003c/li\u003e\n\u003cli\u003eGe, P., Dawson, V.L. \u0026amp; Dawson, T.M. PINK1 and Parkin mitochondrial quality control: a source of regional vulnerability in Parkinson’s disease. \u003cem\u003eMolecular Neurodegeneration\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 20 (2020).\u003c/li\u003e\n\u003cli\u003eBetarbet, R., et al. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. \u003cem\u003eNature Neurosci.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 1301–1306 (2000).\u003c/li\u003e\n\u003cli\u003eCostello, S., Cockburn, M., Bronstein, J., Zhang, X. \u0026amp; Ritz, B. Parkinson's disease and residential exposure to Maneb and Paraquat from agricultural applications in the Central Valley of California. \u003cem\u003eAm. J. Epidemiol.\u003c/em\u003e \u003cb\u003e169\u003c/b\u003e, 919–926 (2009).\u003c/li\u003e\n\u003cli\u003ePaul, K.C., Cockburn, M., Gong, Y., Bronstein, J. \u0026amp; Ritz, B. Agricultural paraquat dichloride use and Parkinson’s disease in California’s Central Valley. \u003cem\u003eInt. J. Epidemiol.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, dyae004 (2024).\u003c/li\u003e\n\u003cli\u003eWong, Y., et al. Neuronal vulnerability in Parkinson disease and putative therapeutics: Should the focus be on axonal and synaptic terminals? \u003cem\u003eMovement Disorders\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 1406–1422 (2019).\u003c/li\u003e\n\u003cli\u003ePacelli, C., et al. Elevated mitochondrial bioenergetics and axonal arborization size are key contributors to the vulnerability of dopamine neurons. \u003cem\u003eCurrent Biol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 2349–2360 (2015).\u003c/li\u003e\n\u003cli\u003eWimalasena, K. The inherent high vulnerability of dopaminergic neurons toward mitochondrial toxins may contribute to the etiology of Parkinson’s disease. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 246–247 (2016).\u003c/li\u003e\n\u003cli\u003eSurmeier, D.J., Obeso, J.A. \u0026amp; Halliday, G.M. Selective neuronal vulnerability in Parkinson disease. \u003cem\u003eNature Reviews Neuroscience\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 101–113 (2017).\u003c/li\u003e\n\u003cli\u003eMasoud, S.T., et al. Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and L-DOPA reversible motor deficits. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 66–75 (2015).\u003c/li\u003e\n\u003cli\u003eBucher, M.L., et al. Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson’s disease. \u003cem\u003eNPJ Parkinsons Dis\u003c/em\u003eease \u003cb\u003e6\u003c/b\u003e, 34 (2020).\u003c/li\u003e\n\u003cli\u003eBurbulla, L.F., et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's disease. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e357\u003c/b\u003e, 1255–1261 (2017).\u003c/li\u003e\n\u003cli\u003eHerrera, A., Muñoz, P., Steinbusch, H.W.M. \u0026amp; Segura-Aguilar, J. Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s Disease? \u003cem\u003eACS Chem. Neurosci.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 702 − 711 (2017).\u003c/li\u003e\n\u003cli\u003eBiosa, A., et al. Dopamine oxidation products as mitochondrial endotoxins, a potential molecular mechanism for preferential neurodegeneration in Parkinson's Disease. \u003cem\u003eACS Chem. Neurosci.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 2849 − 2858 (2018).\u003c/li\u003e\n\u003cli\u003eVauzour, D., Pinto, J.T., Cooper, A.J.L. \u0026amp; Spencer, J.P.E. The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signaling. \u003cem\u003eBiochem. J.\u003c/em\u003e \u003cb\u003e463\u003c/b\u003e, 41–52 (2014).\u003c/li\u003e\n\u003cli\u003eMehta, N.J., et al. Hypochlorite converts cysteinyl-dopamine into a cytotoxic product: A possible factor in Parkinson's disease. \u003cem\u003eFree Radic. Biol. Med.\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e, 44–52 (2016).\u003c/li\u003e\n\u003cli\u003eZhang, F. \u0026amp; Dryhurst, G. Effects of L-cysteine on the oxidation chemistry of dopamine: New reaction pathways of potential relevance to idiopathic Parkinson's disease. \u003cem\u003eJ. Med. Chem.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 1084–1098 (1994).\u003c/li\u003e\n\u003cli\u003eShen, X.M. \u0026amp; Dryhurst, G. Further insights into the influence of L-cysteine on the oxidation chemistry of dopamine: Reaction pathways of potential relevance to Parkinson’s disease. \u003cem\u003eChem. Res. Toxicol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 751–763 (1996).\u003c/li\u003e\n\u003cli\u003eLi, H. \u0026amp; Dryhurst, G. Irreversible inhibition of mitochondrial complex I by 7- (2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): A putative nigral endotoxin of relevance to Parkinson’s disease.\u0026lt;?ColorInfoStart FFFFFF?\u0026gt; \u0026lt;?ColorInfoEnd FFFFFF?\u0026gt;\u003cem\u003eJ. Neurochem.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 1530–1541 (1997).\u003c/li\u003e\n\u003cli\u003eLi, H., Shen, X.M. \u0026amp; Dryhurst, G. Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: Potential relevance to the pathogenesis of Parkinson’s disease. \u003cem\u003eJ. Neurochem.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 2049–2062 (1998).\u003c/li\u003e\n\u003cli\u003eShen, X.M. \u0026amp; Dryhurst, G. Iron- and manganese-catalyzed autoxidation of dopamine in the presence of l-cysteine: Possible insights into iron- and manganese-mediated dopaminergic neurotoxicity. \u003cem\u003eChem. Res. Toxicol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 824–837 (1998).\u003c/li\u003e\n\u003cli\u003eMosca, L., Tempera, I., Lendaro, E., Di Francesco, L. \u0026amp; d’Erme, M. Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells. \u003cem\u003eJ. Neurosci. Res.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, 954–960 (2008).\u003c/li\u003e\n\u003cli\u003eWakamatsu, K., Murase, T., Zucca, F.A., Zecca, L. \u0026amp; Ito, S. Biosynthetic pathway to neuromelanin and its aging process. \u003cem\u003ePigment Cell \u0026amp; Melanoma Research\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 792–803 (2012).\u003c/li\u003e\n\u003cli\u003eMarwah, P.K. Hypochlorite-mediated oxidation of dopamine: Implications in Parkinson’s disease and manganism. Ph.D. dissertation, Wayne State University (2021).\u003c/li\u003e\n\u003cli\u003eNapolitano, A., Di Donato, P. \u0026amp; Prota, G. New regulatory mechanisms in the biosynthesis of pheomelanins: Rearrangement vs. redox exchange reaction routes of a transient 2H-1,4-benzothiazine-o-quinonimine intermediate. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e \u003cb\u003e1475\u003c/b\u003e, 47–54 (2000).\u003c/li\u003e\n\u003cli\u003eNapolitano, A., Memoli, S. \u0026amp; Prota, G. A new insight in the biosynthesis of pheomelanins: Characterization of a labile 1,4-benzothiazine intermediate. \u003cem\u003eJ. Org. Chem.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 3009–3011 (1999).\u003c/li\u003e\n\u003cli\u003eHenrich, M.T., Oertel, W.H., Surmeier, D.J. \u0026amp; Geibl, F.F. Mitochondrial dysfunction in Parkinson’s disease – a key disease hallmark with therapeutic potential. \u003cem\u003eMolecular Neurodegeneration\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 83 (2023).\u003c/li\u003e\n\u003cli\u003eAdetuyi, O.A. \u0026amp; Wimalasena, K. Exposure to Mitochondrial Toxins: An in vitro study of energy depletion and oxidative stress in driving dopaminergic neuronal death in MN9D cells. \u003cem\u003eToxics\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 637 (2025).\u003c/li\u003e\n\u003cli\u003eNagatsu, T., et al. The role of tyrosine hydroxylase as a key player in neuromelanin synthesis and the association of neuromelanin with Parkinson’s disease. \u003cem\u003eJ. Neural Transmission\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 611–625 (2023).\u003c/li\u003e\n\u003cli\u003eRosengren, E., Linder-Eliasson, E. \u0026amp; Carlsson, A. Detection of 5-S-cysteinyldopamine in human brain. \u003cem\u003eJ. Neural Transmission\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e, 247–253 (1985).\u003c/li\u003e\n\u003cli\u003eFornstedt, B., Rosengren, E. \u0026amp; Carlsson, A. Occurrence and distribution of 5-S-cysteinyldopamine, dopa and dopac in the brains of eight mammalian species. \u003cem\u003eNeuropharmacol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 451–454 (1986).\u003c/li\u003e\n\u003cli\u003eFornstedt, B., Bergh, I., Rosengren, E. \u0026amp; Carlsson, A. An improved HPLC-electrochemical detection method for measuring brain levels of 5-S-cysteinyldopamine, 5-S-cysteinyl-3,4-dihydroxyphenylalanine, and 5-S-cysteinyl-3,4-dihydroxyphenylacetic acid. \u003cem\u003eJ. Neurochem.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 578–586 (1990).\u003c/li\u003e\n\u003cli\u003eMalagelada, C. \u0026amp; Greene, L.A. PC12 Cells as a model for parkinson's disease research. In: \u003cem\u003eParkinson’s disease: Molecular and therapeutic insights from model systems.\u003c/em\u003e Edited by R. Nass and S. Przedborski. Cambridge, MA: Academic; p. 375–387 (2008).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Benzothiazine, Cysteinyl-dopamine, Dopamine, Neuromelanin, Paraquat, Parkinson’s disease, Rotenone","lastPublishedDoi":"10.21203/rs.3.rs-9280779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9280779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe fundamental problem in Parkinson\u0026rsquo;s disease is the selective death of dopaminergic neurons in the substantia nigra. This selective death is facilitated by products of dopamine oxidation including a toxin, DHBT-1 (7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2\u003cem\u003eH\u003c/em\u003e-benzo[b][1,4]thiazine-3-carboxylic acid) and a cotoxin, BT-2 (7-(2-aminoethyl)-\u003cem\u003e2H\u003c/em\u003e-benzo[b][1,4]thiazin-5-ol). The cytotoxicity of these compounds was tested both individually and in the presence of the mitochondrial complex I inhibitors rotenone and Paraquat, both of which are implicated as causes of Parkinson\u0026rsquo;s disease. BT-2 potentiates the toxicities of these inhibitors. It also amplifies the toxicity of DHBT-1, reported to also be a complex I inhibitor. Although BT-2 is somewhat unstable, an analogue (2,2-dimethyl-BT-2) is much more stable than BT-2 itself and also enhances the toxicities of DHBT-1, rotenone and Paraquat. We suggest that DHBT-1 and BT-2 are normally detoxified by polymerization into neuromelanin but may accumulate under certain conditions and contribute, in concert with other stresses, to the selective death of dopaminergic neurons.\u003c/p\u003e","manuscriptTitle":"Dopamine-Derived Cotoxins and the Unique Vulnerability of Dopaminergic Neurons in Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-17 20:16:33","doi":"10.21203/rs.3.rs-9280779/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1a51139-7047-42e8-a879-dc71f8a97804","owner":[],"postedDate":"April 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66451920,"name":"Biological sciences/Drug discovery"},{"id":66451921,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-22T00:54:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-17 20:16:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9280779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9280779","identity":"rs-9280779","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}