Aminochrome induces dopaminergic neuronal dysfunction: a new animal model for Parkinson's disease.

L-Dopa continues to be the gold drug in Parkinson's disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson's disease.

Protective and toxic roles of dopamine in Parkinson's disease.

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ-1 (PARK7), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1), serine/threonine-protein kinase (PINK-1), leucine-rich repeat kinase 2 (LRRK2), cation-transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o-quinone, aminochrome and 5,6-indolequinone are formed. However, both dopamine o-quinone and 5,6-indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o-quinone, aminochrome and 5,6-indolequinone polymerization to neuromelanin and (iii) two-electron reduction of aminochrome catalyzed by DT-diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone plays an important role in neurodegeneration in Parkinson's disease since they induce mitochondria and protein degradation dysfunction; formation of neurotoxic alpha synuclein protofibrils and oxidative stress. However, the cells have a protective system against dopamine oxidation composed by dopamine uptake mediated by Vesicular monoaminergic transporter-2 (VMAT-2), neuromelanin formation, two-electron reduction and GSH-conjugation mediated by Glutathione S-transferase M2-2 (GSTM2).

Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity.

We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of ³H-aminochrome 6.3 ± 0.4nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2µM lobeline but not reserpine (1 and 10µM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20µM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11µM) contrasting with a significant decrease in wild type cells (121±11µM, P<0.001); and (iv) a significant decrease in DNA laddering (21 ± 8pixels, P<0.001) cells in comparison with wild type cells treated with 20µM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.

Copper neurotoxicity in rat substantia nigra and striatum is dependent on DT-diaphorase inhibition.

The dependence of copper neurotoxicity on DT-diaphorase inhibition was suggested from results obtained from a cell line derived from substantia nigra. Therefore, the aim of this study was to evaluate whether CuSO4 neurotoxicity in vivo, which was evaluated by determining the contralateral rotation and loss of tyrosine hydroxylase immunostaining, was dependent on DT-diaphorase inhibition by dicoumarol. Animals unilaterally and intranigrally injected with 0.25 nmol of CuSO4 and 2 nmol of dicoumarol presented a significant and characteristic contralateral rotational behavior ( P < 0.01) when they were systemically stimulated with apomorphine (0.5 mg/kg s.c.), similar to that observed in rats injected unilaterally with 6-hydroxydopamine as a positive control. The behavioral effects correlated with the lost of tyrosine hydroxylase-positive staining, since animals unilaterally and intranigrally injected with 0.25 nmol of CuSO4 together with 2 nmol of dicoumarol exhibited extensive loss of tyrosine hydroxylase-positive fiber density in the striatum ( P < 0.01) and cell loss in the substantia nigra ( P < 0.01). Our results support the idea that CuSO4 neurotoxicity is dependent upon DT-diaphorase inhibition.

The catecholaminergic RCSN-3 cell line: a model to study dopamine metabolism.

RCSN-3 cells are a cloned cell line derived from the substantia nigra of an adult rat. The cell line grows in monolayer and does not require differentiation to express catecholaminergic traits, such as (i) tyrosine hydroxylase; (ii) dopamine release; (iii) dopamine transport; (iv) norepinephrine transport; (v) monoamine oxidase (MAO)-A expression, but not MAO-B; (vi) formation of neuromelanin; (vii) VMAT-2 expression. In addition, this cell line expresses serotonin transporters, divalent metal transporter, DMT1, dopamine receptor 1 mRNA under proliferating conditions, and dopamine receptor 5 mRNA after incubation with dopamine or dicoumarol. Expression of dopamine receptors D(2), D(3) and D(4) mRNA were not detected in proliferating cells or when the cells were treated with dopamine, CuSO(4), dicoumarol or dopamine-copper complex. Angiotensin II receptor mRNA was also found to be expressed, but it underwent down regulation in the presence of aminochrome. Total quinone reductase activity corresponded 94% to DT-diaphorase. The cells also express antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. This cell line is a suitable in vitro model for studies of dopamine metabolism, since under proliferating conditions the cells express all the pertinent markers.

Aminochrome as a preclinical experimental model to study degeneration of dopaminergic neurons in Parkinson's disease.

Four decades after L-dopa introduction to PD therapy, the cause of Parkinson's disease (PD) remains unknown despite the intensive research and the discovery of a number of gene mutations and deletions in the pathogenesis of familial PD. Different model neurotoxins have been used as preclinical experimental models to study the neurodegenerative process in PD, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone. The lack of success in identifying the molecular mechanism for the degenerative process in PD opens the question whether the current preclinical experimental models are suitable to understand the degeneration of neuromelanin-containing dopaminergic neurons in PD. We propose aminochrome as a model neurotoxin to study the neurodegenerative processes occurring in neuromelanin-containing dopaminergic neurons in PD. Aminochrome is an endogenous compound formed during dopamine oxidation and it is the precursor of neuromelanin, a substance whose formation is a normal process in mesencephalic dopaminergic neurons. However, aminochrome itself can induce neurotoxicity under certain aberrant conditions such as (i) one-electron reduction of aminochrome catalyzed by flavoenzymes to leukoaminochrome o-semiquinone radical, which is a highly reactive neurotoxin; or (ii) the formation of aminochrome adducts with alpha-synuclein, enhancing and stabilizing the formation of neurotoxic protofibrils. These two neurotoxic pathways of aminochrome are prevented by DT-diaphorase, an enzyme that effectively reduces aminochrome with two-electrons preventing both aminochrome one-electron reduction or formation alpha synuclein protofibrils. We propose to use aminochrome as a preclinical experimental model to study the neurodegenerative process of neuromelanin containing dopaminergic neurons in PD.

Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease.

There are several interrelated mechanisms involving iron, dopamine, and neuromelanin in neurons. Neuromelanin accumulates during aging and is the catecholamine-derived pigment of the dopamine neurons of the substantia nigra and norepinephrine neurons of the locus coeruleus, the two neuronal populations most targeted in Parkinson's disease. Many cellular redox reactions rely on iron, however an altered distribution of reactive iron is cytotoxic. In fact, increased levels of iron in the brain of Parkinson's disease patients are present. Dopamine accumulation can induce neuronal death; however, excess dopamine can be removed by converting it into a stable compound like neuromelanin, and this process rescues the cell. Interestingly, the main iron compound in dopamine and norepinephrine neurons is the neuromelanin-iron complex, since neuromelanin is an effective metal chelator. Neuromelanin serves to trap iron and provide neuronal protection from oxidative stress. This equilibrium between iron, dopamine, and neuromelanin is crucial for cell homeostasis and in some cellular circumstances can be disrupted. Indeed, when neuromelanin-containing organelles accumulate high load of toxins and iron during aging a neurodegenerative process can be triggered. In addition, neuromelanin released by degenerating neurons activates microglia and the latter cause neurons death with further release of neuromelanin, then starting a self-propelling mechanism of neuroinflammation and neurodegeneration. Considering the above issues, age-related accumulation of neuromelanin in dopamine neurons shows an interesting link between aging and neurodegeneration.

Corrigendum: Commentary: Evaluation of Models of Parkinson's Disease.

[This corrects the article on p. 161 in vol. 10, PMID: 27147953.].

Aminochrome as New Preclinical Model to Find New Pharmacological Treatment that Stop the Development of Parkinson's Disease.

The pharmacological treatment of Parkinson's disease (PD) is limited to dopamine agonists and anti-cholinergic drugs that do not stop the progress of disease. LDopa was introduced to the treatment in 1967; this drug is still the best and most commonly used drug since it generates a real improvement in patient quality of life, but the disadvantage of L-dopa is that this positive effect is followed by severe side effects such as dyskinesia. The search for a new drug in the treatment of PD is limited to compounds which decrease the side effects of the drugs used in the treatment of the disease, such as L-dopa-induced dyskinesia. One possible explanation for pharmaceutical companies not developing new drugs to stop disease development is because the mechanism which induces the loss of dopaminergic neurons containing neuromelanin of the nigrostriatal system is still unknown. The discovery of genes (alpha-synuclein, parkin, pink-1, DJ- 1, LRRK2, GBA1, etc.) associated with familial forms of PD resulted in an enormous input into basic research in order to understand the role of these proteins in the disease. It is generally accepted that the loss of dopaminergic neurons containing neuromelanin involves mitochondrial dysfunction, protein degradation dysfunction, the aggregation of alpha-synuclein to neurotoxic oligomers, oxidative neuroinflammation and endoplasmic reticulum stress, but the question of what induces these mechanisms remains unanswered. Aminochrome, the product of dopamine oxidation and the precursor of neuromelanin, is directly involved in five of the six mechanisms and may be a better PD preclinical model.

DT-diaphorase protects astrocytes from aminochrome-induced toxicity.

Astrocytes are exposed to aminochrome via the oxidation of dopamine that is taken up from the synaptic cleft after its release from dopaminergic neurons. Glutathione transferase M2-2 (GSTM2) has been shown to protect astrocytes from aminochrome-induced toxicity, but astrocytes also express DT-diaphorase, which has been shown to prevent aminochrome-induced neurotoxicity in dopaminergic neurons. Therefore, the question is whether DT-diaphorase also protects astrocytes from aminochrome-induced toxicity. DT-diaphorase is constitutively expressed in U373MG cells, and its inhibition by dicoumarol induced a significant increase of aminochrome-induced cell death. However, the inhibition of DT-diaphorase in U373MGsiGST6 cells, which have 74% of GSTM2 gene expression silenced, resulted in a more than 2-fold increase in cell death, suggesting that DT-diaphorase plays an important role in preventing aminochrome-induced toxicity in astrocytes.

Aminochrome Toxicity is Mediated by Inhibition of Microtubules Polymerization Through the Formation of Adducts with Tubulin.

In this study, we investigated the role of adducts formation between aminochrome and tubulin and its interference in microtubules assembly and stability in aminochrome-induced toxicity in SH-SY5Y cells. We also investigated whether changes in the microtubules structures are an early event that could affect tubulin expression. We demonstrated in vitro that aminochrome tubulin adducts inhibit tubulin polymerization and that aminochrome induces microtubules disassembly. Moreover, when the SH-SY5Y cells were incubated with aminochrome, we observed an increase in soluble tubulin, indicating depolymerization of microtubules. Aminochrome generates disruption of the microtubules network, leading to changes in the morphology of the cells inducing cell death, in a dose- and time-dependent manner. Interestingly, these changes preceded cell death and were partly inhibited by paclitaxel, a microtubule-stabilizing agent. Furthermore, we observed that aminochrome increased early tubulin expression before significant cell death occurred. Consequently, all these antecedents suggest that aminochrome toxicity is mediated by early disruption of microtubules network, where the adduct formation between aminochrome and tubulin could be responsible for the inhibition in the assembly microtubules and the loss of microtubules stability. Possibly, the early changes in tubulin expression could correspond to compensatory mechanisms against the toxic effects of aminochrome.

Impact of Plant-Derived Flavonoids on Neurodegenerative Diseases.

Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation of astrocytes and microglia, astrocyte associated protection of neuronal progenitor cells, neuronal differentiation and neuritogenesis. It was observed the flavonoids also induced neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells. Moreover, with the objective of seeking preclinical pharmacological evidence of these molecules, in order to assess its future use in the treatment of neurodegenerative disorders, we have evaluated the effects of flavonoids in preclinical in vitro models of neuroinflammation associated with Parkinson's disease and glutamate toxicity associated with ischemia. In particular, our efforts have been directed to identify mechanisms involved in the changes in viability, morphology, and glial cell function induced by flavonoids in cultures of glial cells and neuronal cells alone or in interactions and clarify the relation with their neuroprotective and morphogetic effects.

Glutathione transferase-M2-2 secreted from glioblastoma cell protects SH-SY5Y cells from aminochrome neurotoxicity.

U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 µM aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of (3)H-aminochrome into U373MG cells was significantly reduced in the presence of 2 µM nomifensine (P < 0.001) 100 µM imipramine (P < 0.001) and 50 mM dopamine (P < 0.001). Interestingly, U373MG cells excrete GSTM2 into the conditioned medium and the excretion was significantly increased (2.7-fold; P < 0.001) when the cells were pretreated with 50 µM aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 µM aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of (14)C-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.

DT-Diaphorase Prevents Aminochrome-Induced Alpha-Synuclein Oligomer Formation and Neurotoxicity.

It was reported that aminochrome induces the formation of alpha synuclein (SNCA) oligomers during dopamine oxidation. We found that DT-diaphorase (NQO1) prevents the formation of SNCA oligomers in the presence of aminochrome determined by Western blot, transmission electron microscopy, circular dichroism, and thioflavin T fluorescence, suggesting a protective role of NQO1 by preventing the formation of SNCA oligomers in dopaminergic neurons. In order to test NQO1 protective role in SNCA neurotoxicity in cellular model, we overexpressed SNCA in both RCSN-3 cells (wild-type) and RCSN-3Nq7 cells, which have constitutive expression of a siRNA against NQO1. The expression of SNCA in RCSN-3SNCA and RCSN-3Nq7SNCA cells increased 4.2- and 4.4-fold, respectively. The overexpression of SNCA in RCSN-3Nq7SNCA cells induces a significant increase in cell death of 2.8- and 3.2-fold when they were incubated with 50 and 70 µM aminochrome, respectively. The cell death was found to be of apoptotic character determined by annexin/propidium iodide technique with flow cytometry and DNA laddering. A Western blot demonstrated that SNCA in RCSN-3SNCA is only found in monomer form both in the presence of 20 µM aminochrome or cell culture medium contrasting with RCSN-3Nq7SNCA cells where the majority SNCA is found as oligomer. The antioligomer compound scyllo-inositol induced a significant decrease in aminochrome-induced cell death in RCSN-3Nq7SNCA cells in comparison to cells incubated in the absence of scyllo-inositol. Our results suggest that NQO1 seems to play an important role in the prevention of aminochrome-induced SNCA oligomer formation and SNCA oligomers neurotoxicity in dopaminergic neurons.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A 1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A 1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

One-electron reduction of 6-hydroxydopamine quinone is essential in 6-hydroxydopamine neurotoxicity.

6-Hydroxydamine has widely been used as neurotoxin in preclinical studies related on the neurodegenerative process of dopaminergic neurons in Parkinson's disease based on its ability to be neurotoxic as a consequence of free radical formation during its auto-oxidation to topaminequinone. We report that 50-µM 6-hydroxydopamine is not neurotoxic in RCSN-3 cells derived from substantia nigra incubated during 24 h contrasting with a significant sixfold increase in cell death (16 ± 2 %; P < 0.001) was observed in RCSN-3NQ7 cells expressing a siRNA against DT-diaphorase that silence the enzyme expression. To observe a significant cell death in RCSN-3 cells induced by 6-hydroxydopamine (24 ± 1 %; P < 0.01), we have to increase the concentration to 250 µm while a 45 ± 2 % cell death (P < 0.001) was observed at this concentration in RCSN-3NQ7 cells. The cell death induced by 6-hydroxydopamine in RCSN-3NQ7 cells was accompanied with a (i) significant increase in oxygen consumption (P < 0.01), (ii) depletion of reduced glutathione and (iii) a significant decrease in ATP level (P < 0.05) in comparison with RCSN-3 cells. In conclusion, our results suggest that one-electron reduction of 6-hydroxydopamine quinone seems to be the main reaction responsible for 6-hydroxydopamine neurotoxic effects in dopaminergic neurons and DT-diaphorase seems to play an important neuroprotective role by preventing one-electron reduction of topaminequinone.

Dopamine oxidation and autophagy.

The molecular mechanisms involved in the neurodegenerative process of Parkinson's disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinson's disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i) the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii) the formation of adducts with α- and ß-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.

Protective effects of nicotine against aminochrome-induced toxicity in substantia nigra derived cells: implications for Parkinson's disease.

Parkinson's disease is a debilitating progressive neurodegenerative disorder that results from the loss of or damage to dopaminergic cells containing neuromelanin in the substantia nigra (SN). The underlying neurodegenerative mechanism(s), however, remain elusive. Aminochrome, the precursor of neuromelanin is an endogenous substance capable of inducing selective neurotoxicity to dopaminergic neurons in SN. Nicotine, on the other hand, may offer protective effects against dopaminergic cell damage induced by various neurotoxins including MPTP and salsolinol. In this study, we sought to determine whether nicotine may also protect against aminochrome-induced toxicity in SN derived RCSN-3 cells. Exposure of RCSN-3 cells to a combination of aminochrome (50 µM) and dicoumarol (50 µM) for 48 h induced approximately 70 % cell death. Pretreatment with nicotine, dose-dependently blocked this toxicity. The effects of nicotine in turn were dose-dependently blocked by mecamylamine, a non-selective nicotinic receptor antagonist. These results suggest involvement of nicotinic receptors in protective effects of nicotine against aminochrome-induced toxicity and provide further evidence for possible therapeutic effects of nicotine or nicotinic agonists in Parkinson's disease.