Xue-Ming Shen

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

Abstract: We have proposed that a very early step in the pathogenesis of idiopathic Parkinsons disease is the elevated translocation of l‐cysteine into neuromelanin‐pigmented dopaminergic neurons in the substantia nigra. This influx of l‐cysteine was proposed to divert the normal neuromelanin pathway by scavenging dopamine‐o‐quinone, formed by autoxidation of cytoplasmic dopamine, to give initially 5‐S‐cysteinyldopamine, which is further oxidized to 7 ‐ (2 ‐ aminoethyl) ‐ 3,4 ‐ dihydro ‐ 5 ‐ hydroxy ‐ 2H ‐ 1,4 ‐ benzothiazine‐3‐carboxylic acid (DHBT‐1). In a recent report, it was demonstrated that DHBT‐1 evokes inhibition of complex I respiration when incubated with intact rat brain mitochondria and a time‐dependent irreversible inhibition of NADH‐coenzyme Q1 (CoQ1) reductase when incubated with mitochondrial membranes. In this study, it is established that the time dependence of NADH‐CoQ1 reductase inhibition reflects the oxidation of DHBT‐1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an o‐quinone imine intermediate that rearranges to 7‐(2‐aminoethyl) ‐ 5 ‐ hydroxy ‐ 1,4 ‐ benzothiazine ‐ 3 ‐ carboxylic acid (BT‐1) and decarboxylates to 7‐(2‐aminoethyl)‐5‐hydroxy‐1,4‐benzothiazine (BT‐2), which are further catalytically oxidized to o‐quinone imine intermediates. The electrophilic o‐quinone imine intermediates formed in these mitochondria‐catalyzed oxidations of DHBT‐1, BT‐1, and BT‐2 are proposed to bind covalently to key sulfhydryl residues at the complex I site, thus evoking irreversible inhibition of NADH‐CoQ1 reductase. Evidence for this mechanism derives from the fact that greater than equimolar concentrations of glutathione completely block inhibition of NADH‐CoQ1 reductase by DHBT‐1, BT‐1, and BT‐2 by scavenging their electrophilic o‐quinone imine metabolites to form glutathionyl conjugates. The results of this investigation may provide insights into the irreversible loss of glutathione and decreased mitochondrial complex I activity, which are both anatomically specific to the substantia nigra and exclusive to Parkinsons disease.

Oxidative metabolites of 5-S-cysteinyldopamine inhibit the α-ketoglutarate dehydrogenase complex: possible relevance to the pathogenesis of Parkinson's disease

Summary. A characteristic change in the substantia nigra of Parkinsons disease patients is an apparent accelerated rate of dopamine oxidation as evidenced by an increased 5-S-cysteinyldopamine (5-S-CyS-DA) to dopamine ratio. However, 5-S-CyS-DA is more easily oxidized than dopamine to give 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). Previous studies have demonstrated that DHBT-1 can be accumulated by intact rat brain mitochondria and inhibits complex I but not complex II respiration. In this study, it is shown that DHBT-1 also inhibits the α-ketoglutarate dehydrogenase complex (α-KGDH) but not cytochrome c oxidase (complex IV). The inhibition of α-KGDH is dependent on the oxidation of DHBT-1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an electrophilic o-quinone imine that covalently modifies active site sulfhydryl residues. The latter conclusion is based on the ability of ≧ equimolar glutathione to block the inhibition of α-KGDH by DHBT-1, without altering its rate of mitochondrial membrane-catalyzed oxidation, by scavenging the electrophilic o-quinone intermediate forming glutathionyl conjugates which have been isolated and spectroscopically characterized. Activities of mitochondrial α-KGDH and complex I, but not other respiratory complexes, are decreased in the parkinsonian substantia nigra. Such changes together with evidence for accelerated dopamine oxidation, increased formation of 5-S-CyS-DA and the ease of oxidation of this conjugate to DHBT-1 which inhibits α-KGDH and complex I, without affecting other respiratory enzyme complexes, suggests that the latter putative metabolite might be an endotoxin that contributes to the α-KGDH and complex I defects in Parkinsons disease.

Influence of glutathione on the oxidation chemistry of 5-S-cysteinyldopamine: potentially neuroprotective reactions of relevance to Parkinson's disease

Abstract In recent reports from this laboratory we have hypothesized that a key step underlying the degeneration of pigmented dopaminergic neurons in the substantia nigra pars compacta (SN c ) in Parkinsons disease is an accelerated rate of oxidation of intraneuronal dopamine in the presence of l -cysteine (CySH) to form initially 5- S -cysteinyldopamine (5- S -CyS-DA). 5- S -CyS-DA, however, is more easily oxidized than dopamine in a reaction which leads to the dihydrobenzothiazine (DHBT) 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2 H -1,4-benzothiazine-3-carboxylic acid (DHBT-1), a putative endogenously-formed metabolite that may be responsible for inhibition of mitochondrial complex I and α-ketoglutarate dehydrogenase, characteristic defects in the parkinsonian SN c . In this investigation it is demonstrated that glutathione (GSH) dramatically attenuates the oxidative transformation of 5- S -CyS-DA into DHBT-1 by two major pathways. In one pathway GSH displaces the cysteinyl residue from the o -quinone proximate oxidation product of 5- S -CyS-DA forming the corresponding glutathionyl conjugate that is attacked by GSH, to form 2,5-di- S -glutathionyldopamine, or by released CySH to give 2- S -cysteinyl-5- S -glutathionyldopamine. The former is the precursor of 2,5,6-tris- S -glutathionyldopamine, a major reaction product. However, intramolecular cyclization of the o -quinone proximate product of 2- S -cysteinyl-5- S -glutathionyldopamine is the first step in a pathway leading to glutathionyl conjugates of 8-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2 H -1,4-benzothiazine-3-carboxylic acid (DHBT-5). The second pathway involves nucleophilic addition of GSH to the o -quinone proximate oxidation product of 5- S -CyS-DA forming 2- S -glutathionyl-5- S -cysteinyldopamine the precursor of a number of glutathionyl conjugates of DHBT-1. These results raise the possibility that strategies which elevate intraneuronal levels of GSH in dopaminergic SN c cells in Parkinsons disease patients may block formation of the putative mitochondrial toxin DHBT-1 and hence be neuroprotective.

Potential Roles of Accelerated Dopamine Oxidation, Altered Glutathione Metabolism, 5-S-Cysteinyl-Dopamine and its Oxidative Metabolites in the Pathogenesis of Parkinson’s Disease

Many factors appear to be involved in the neurotoxic mechanism that underlies the degeneration of neuromelanin-pigmented dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson’s disease (PD). These include: (1) a massive loss of glutathione (GSH) without corresponding increases of glutathione disulfide, a very early change in the parkinsonian SNpc; (2) increased intraneuronal superoxide (O 2 -· ) generation; (3) mobilization of low molecular weight iron from storage proteins, part of which is scavenged by neuromelanin; (4) increased activity of γ-glutamyl transpeptidase; (5) an accelerated rate of dopamine (DA) oxidation that does not lead to increased neuromelanin deposition but rather increased formation of 5-S-cysteinyldopamine (5-S-CyS-DA); and (6) decreased activities of mitochondrial complex I and α-ketoglutarate dehydrogenase (α-KGDH). Based on these changes and information derived from studies of the dopaminergic neurotoxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and methamphetamine, a new pathological mechanism is proposed that might contribute to an understanding of SNpc cell death in PD. This neurotoxic mechanism is suggested to be initiated by a transient dopaminergic neuron energy impairment that triggers a cascade of processes which ultimately result in the intraneuronal oxidation of DA by O 2 -· in the presence of cysteine to form 5-S-CyS-DA. Further O 2 -· mediated oxidation of 5-S-CyS-DA is proposed to lead to aberrant dihydrobenzothiazine and benzothiazine metabolites responsible for complex I and α-KGDH inhibition.