Mitochondrial toxins and neurodegenerative diseases

[Frontiers in Bioscience 12, 986-1007, January 1, 2007] 986 Mitochondrial toxins and neurodegenerative diseases Antonio Ayala, Jose L. Venero, Josefina Cano and Alberto Machado Departamento de Bioquimica, Bromatologia, Toxicologia y Medicina Legal. Facultad de Farmacia University of Seville, Spain TABLE OF CONTENTS 1. Abstract 2. Mitochondrial toxin involved in neurodegeneration 3. Toxin that produced a neurodegeneration like that found in parkinson’s disease 3.1. 1. Methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) 3.2. Tetrahydroisoquinoline derivatives as possible endogenous neurotoxins 3.2.1. 1. Methyl-1,2,3,4-tetrahydroisoquinoline as neuroprotector of dopaminergic systems 3.3. Rotenone 3.3.1. In vivo rotenone models of Parkinson’s disease 3.3.2. Rotenone and oxidative stress 3.3.3. Rotenone and the proteasome. 3.3.4. Rotenone and apoptosis 3.3.5. Rotenone and the dopaminergic system 3.4. Other inhibitors of Complex I 4. Toxins that produced a neurodegeneration like that found in Huntington disease: Inhibitor of succinate dehydrogenase 4.1. Pathways of cells death induced by 3-NPA. 4.1.1. Energy depletion 4.1.2. Excitotoxicity 4.1.3. Oxidative stress 4.2. Specificity of the 3-NPA toxicity. 4.3. CNS regional vulnerability to toxins 4.4. Mechanism of neuronal cell death 4.5. Other inhibitors of complex II 5. Conclusions 6. Perspective 7. Aknowlegments 8. References 1. ABSTRACT The selective loss of a particular subset of neurons is a common feature of neurodegenerative disorders. A failure in respiratory chain complex activities in mitochondria seems to be a causative factor. The aim of this review is to describe the most important toxins affecting the mitochondrial function, which could be involved in the incidence of some of these diseases: MPTP, rotenone and 3-nitropropionic (3-NPA). 2. MITOCHONDRIAL TOXIN INVOLVED IN NEURODEGENERATION Neurodegenerative disorders are characterized clinically by insidious onset and a slow progressive course. Pathologically, these diseases share a common feature: the selective loss of a particular subset of neurons for unknown reasons, such as cerebral cortical neurons in Alzheimer’s disease (AD), substantia nigra neurons in Parkinson’s disease (PD), spinal motoneurons in Amyotrophic Lateral Sclerosis (ALS), and medium-sized striatal neurons in Huntington’s disease (HD). Like many other age-related diseases, they are frequently hereditary, showing some familial association. Therefore, a genetic basis can be assumed. However, their etiology is to a large extent unknown. Metabolic defects, produced by failure in respiratory chain complex activities in mitochondria, are thought to underlie defects in energy metabolism and seem to be a causative factor in several of these neurodegenerative diseases (1,2). Evidence for mitochondria being a damage site in neurodegenerative disorders is partially based on the impairments in respiratory function found in some of these diseases. Small subsets of PD patients have hereditary characteristics including Mendelian pedigrees. There are different kinds of PD and some of them are better known than others: one is related to the gen encoding PINK1 (PTEN-induced kinase) protein, a kinase localized in mitochondria (3); another is related to the gen encoding parkin, a E3 ubiquitin ligase (4); and a rare autosomal dominant form due to mutations in alpha-synuclein (5). In these cases, mitochondrial dysfunction has been described. Mitochondria and neurodegenerative diseases 987 Genetic inactivation of parkin in mice resulted in nigrostriatal defects associated with a decrease in several mitochondrial proteins including some subunits of pyruvate dehydrogenase, complex I and complex IV with a general decrease in mitochondrial state 3 respiration. Expression of alpha-synuclein mutants in various cell systems has resulted in altered mitochondrial function (6-8). Moreover, in idiopathic PD patients, reduced complex I activity was found in autopsied substantia nigra (9-12) and in platelets (13,14). Similarly, early onset of familial AD has been associated with mutations in the Abeta precursor protein (APP) and the presenilin-containing APP peptide processing complexes (15). Abeta peptide toxicity might also be related to mitochondrial dysfunction. The Abeta peptide has been reported entering the mitochondria, binding to mitochondrial alcohol dehydrogenase, and increasing mitochondrial ROS production (16,17). In addition, incubation of rat brain mitochondria with the Abeta 25–35 fragment results in a selective and dose independent inhibition of complex IV (18). Mitochondrial oxidative phosphorylation defects have also been detected in a variety of tissues from late-onset AD patients (19,20). Moreover, in familial cases, ALS is caused by mutations in the Cu,Zn-SOD (Sod1) (21). Transgenic mice that express a human mutant Cu,Zn-SOD develop a motor neuron disease associated with vacuolated mitochondria containing high concentrations of Cu,Zn-SOD (22). Mitochondrial dysfunction has also been implicated in the etiology of Huntington disease (23,24); mtDNA rearrangements are elevated in the basal ganglia of patients with this disease (25). Recently, Wallace (26) has pointed out that these age-related degenerative diseases are increasing to epidemic proportions in all industrialized countries. Today, AD affects about 4.5 million Americans, and calculations say it will reach 11-16 million by 2050 (27). Parkinson’s disease afflicts approximately one million persons in the United States today, 60.000 new cases are diagnosed every year, and the incidence is expected to quadruple by 2040 (28). Wallace (29) also indicated that the nature and frequency of genetic variants in the human population has not changed significantly in the past 50 years, even though the incidence of these diseases has climbed continuously. Mitochondria are the only human genetic system that embodies the features necessary to explain common features observed in these age related diseases. Environment seems to be the most important factor in continuous change, diet being the most dynamic change. One of the most important factors included in both environment and diet is the presence of many toxins, some of them producing mitochondrial inhibition. These toxins could be involved in the incidence increase of some of these degenerative diseases; environmental toxins and lack of dietary vitamin E have also been suggested as causing ALS disease (30,31). In this review our aim is to describe the most important toxins affecting the mitochondrial function, which could be involved in the incidence of some neurodegenerative diseases. Animal models were used for studying these diseases. 3. TOXIN THAT PRODUCED A NEURODEGENERATION LIKE THAT FOUND IN PARKINSON’S DISEASE 3.1. 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) Parkinson’s disease is characterized by the progressive loss of dopamine (DA) in the caudate nucleus, putamen, and substantia nigra (SN) upon the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), resulting in cardinal motor symptoms such as tremor at rest, bradikinesia, muscular rigidity, stooped posture, and instability. PD is believed to be caused both by genetic and environmental factors (32). However, and in spite of the fact that familial PD correlates with mutations of genes that encode several proteins, including alpha-synuclein, parkin and ubiquitin C terminal hydrolase-L1 (33), the etiology of idiopathic PD, which accounts for more than 90% of PD, is still not fully understood. It seems to be multifactorial (34- 36). It is well documented that there is an epidemiological link between PD and people who are associated with rural living and working in agriculture. In particular, people exposed to various herbicides and insecticides in an agricultural setting showed an increased risk of developing PD (37). The possibility that environmental neurotoxins played an important role in PD came to light in the early 1980s when several drug users in Northern California developed an acute state of akinesia (initially confused with catatonia) following the intravenous injection of a street preparation of 1-methyl-4-phenyl-4-propionpiperidine (MPPP), an analog of the narcotic meperidine. Also, the finding that 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), which was inadvertently produced during the illicit synthesis of MPPP led to Parkinsonism (38) shifted the focus toward environmental factors as potential PD initiators or contributors. The administration of MPTP produces extensive destruction of nigrostriatal dopaminergic neurons. Indeed, this compound is able to produce a severe parkinsonian-like syndrome in humans and in primates (38,39). The neurotoxic role of MPTP is critically dependent on its metabolite MPP+, formed in glial cells in the reaction catalyzed by the mitochondrial monoamino oxidase B (MAOB). MPP+ then accumulates inside the mitochondria, where it binds to Complex I (40-44) causing an inhibition of NAD-linked mitochondrial respiration. Human mitochondrial complex I (NADH:ubiquinone oxidoreductase; EC: 1.6.5.3) is a large multisubunit assembly comprising 39 nuclear encoded and 7 mitochondrially encoded subunits (45). Therefore, the cellular damage caused by MPP+ is due primarily to energy depletion caused by the specific binding to Complex I (46). This may then lead to neuronal depolarization and secondary excitotoxicity (47). A consequence of this is an influx of Ca2+ into neurons, which accumulates in mitochondria. Mitochondria and neurodegenerative diseases 988 A secondary effect of complex I inhibition is the damage caused by free radical production (48-51). O2 - formation by Complex I appears to be the primary source of O2 - in the brain under normal conditions and in a variety of pathological scenarios ranging from aging to Parkinson's disease (48,52). During electron transfer, electrons from NADH are passed to ubiquinone in a quinone-reducing site via the flavin (FMN) and iron-sulfur centers (49). Ubisemiquinone (UQ10) species generated in the course of electron transfer reactions in the respiratory chain donate electrons to O2 and provides a constant source of O2 - (53- 55). The resulting semiquinone can also be reduced by an electron to form QH2. But in the presence of complex I inhibitors such as rotenone and piericidin this reduction step is strongly slowed, so the semiquinone accumulates and O2 - production increases (56,57). Inhibition of mitochondrial complex I opens mitochondrial permeability transition pore and may trigger apoptotic cell death (32). Inhibition of complex I creates an environment of oxidative stress that ultimately leads to aggregation of alpha-synuclein and the subsequent death of DA neurons (56). It also makes neurons vulnerable to glutamate excitotoxicity. All these results show that impairments in complex I may be central to the pathogenesis of DA neuronal demise in sporadic PD (59). Reduced complex I activity may predispose to excitotoxicity by altering ATP levels, by impairing Ca2+ homeostasis, or both (60). Reduced ATP levels decrease the activity of the plasma membrane Na+/K+ ATPase, resulting in partial neuronal depolarization. Neuronal depolarization decreases the voltage dependent Mg2+ blockade of the N-methyl- D-aspartate (NMDA) glutamate receptor. Under these conditions, even normal levels of extracellular glutamate may cause excitotoxic activation of NMDA receptors and elevation of intracellular Ca2+. Moreover, chronic complex I defects have been shown to disrupt normal Ca2+ signalling in neural cells (61). The most important argument against the animal model of PD produced by the toxicity of MPTP is that it was unable to reproduce the neuronal inclusions called Lewy bodies described in PD (62,63). However, recently Fornai et al. (64) have developed a mouse Parkinson’s disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease, severe striatal dopamine depletion, nigral cell loss along with the formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, this model also caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein dependent manner supporting the probable implication of toxicity in parkinsonism. 3.2. Tetrahydroisoquinoline derivatives as possible endogenous neurotoxins Since the discovery of MPTP toxicity particular attention has been focused on the possible role of different structurally MPTP-like endogenous amines. Two of the more significant families of compounds with specific neurotoxic effects on the dopaminergic system producing parkinsonism in experimental animals, as does MPTP, are the tetrahydroisoquinolines (TIQs) and beta-carbolines (65,66). Beta-carbolines have structures similar to those of MPTP/MPP+, and may be synthesized in vivo from tryptophan via tryptamine (32). Like MPTP, betacarbolines may be precursor neurotoxins that are N- methylated and oxidized by MAO-B to form, in their case, beta-carbolinium ions, which may produce neuronal death and PD symptoms. Various TIQs have also been found in both normal and PD brains as endogenous amines by gas chromatography-mass spectrometry (32). TIQ, 1MeTIQ, N- Me-TIQ, N-Me-6,7-(OH)2-TIQ (N-Me-norsalsolinol), 1,N- (Me)2-6,7-(OH)2-TIQ (N-Me-salsolinol), 1-phenyl-TIQ, N- Me-1-phenyl-TIQ, and 1-benzyl-TIQ (1-Bn-TIQ). Isoquinoline derivatives have been found in foods (67). 1,2,3,4-Tetrahydroisoquinoline is present in flour (0.52 ng/g), banana (2.2 ng/g), cheese (5.2 ng/g), broiled sardine (0.96 ng/g), broiled beef (1.3 ng/g), and the yolk (1.8 ng/g) and white (2.2 ng/g) of boiled eggs and in various alcoholic beverages. They are produced in the CNS by condensation of biogenic amines (e.g. catecholamines and phenylethylamines) with aldehydes (or with alpha-keto acids followed by decarboxylation) by the Picket-Spengler reaction. Isoquinoline derivatives are metabolized by various enzymes including N-methyltransferase and MAO (but very slow compared with MPTP) to produce N- methylated derivatives and isoquinolinium cations, respectively (68,69). Isoquinoline derivatives are also metabolized in brain and liver by cytochrome P450 isoenzymes to produce 4-hydroxylated products (70). Physiologically, these compounds may be involved in the regulation of monoamine function through reversible and competitive inhibition of enzymes involved in monoamine synthesis and metabolism (e.g. tyrosine hydroxylase, l-aromatic amino acid decarboxylase, catechol-O-methyltransferase, and MAO-A and -B) (65). Their methylation in the reaction catalyzed by N- methyltransferase and oxidation with MAO participation leads, similarly as in the case of MPTP, to the formation of N-methylisoquinoline ions, exhibiting neurotoxic properties, which however are much weaker (71). Tetrahydroisoquinolines are complex I inhibitors after their transformation into N-methylisoquinoline ions. Neurotoxic effects and inhibitory influence on the activity of mitochondria are attributed mostly to N- methylisoquinoline ions, whose formation, contrary to MPTP, requires initial N-methylation, catalyzed by N- methyltransferase, in addition to MAOB action. Low availability of this enzyme can explain much lower neurotoxicity of tetrahydroisoquinolines (72) in comparison with MPTP. Whether a relationship exists between the structure and the potency to inhibit mitochondrial respiration and complex I has been studied. Presence of a Mitochondria and neurodegenerative diseases 989 phenyl ring at or near the C1 position augmented the inhibitory potency against NADH-linked respiration and complex I activity (71). N-methylation had little effect on the inhibition of NADH-linked respiration and complex I activity; however, oxidation of N-methylated isoquinoline into N-methylisoquinolinium ion augmented the potency to inhibit NADH-linked respiration. The order of potency of the isoquinoline derivatives is isoquinolinium cations > isoquinolines > dihydroisoquinolines > 1,2,3,4- tetrahydroisoquinolines showing that pyridinium cations are more potent than their corresponding neutral congeners in inhibiting mitochondrial respiration (73). Experimental studies indicated that isoquinolines (e.g. N-methylsalsolinol) could initiate apoptic process in dopaminergic neurons by the generation of free radicals. Isoquinoline and 1,2,3,4-tetrahydroisoquinoline induced apoptotic cell death in PC12 cells and SK-N-MC dopaminergic cell lines by a mechanism involving generation of free radicals, perhaps secondary to inhibition of the mitochondrial respiratory chain (74). These endogenous and exogenous heterocyclic molecules, which are structurally related to MPTP/ MPP+, such as isoquinolines and beta-carbolines, also have the specific similar toxic properties on DA cells, which are also conferred by their uptake by the DAT (44). This shows that DAT might be responsible for the selectivity of DA cell death in these PD models. 3.2.1. 1-methyl-1,2,3,4-tetrahydroisoquinoline as neuroprotector of dopaminergic systems Other isoquinolines, e.g. 1-methyl-1,2,3,4- tetrahydroisoquinoline (1Me-TIQ) can exhibit neuroprotective activity. This compound differs in one methyl group from TIQ. The amount of TIQ seems to increase in the Parkinson brain (75). It is toxic to nigro-striatal dopaminergic neurons (76). However, 1MeTIQ decreases in Parkinson's disease and also seems to decrease with age in humans (77). An occurrence of TIQ and MeTIQ has been reported in two areas especially influenced by aging and Parkinson's disease-substantia nigra (SN) and striatum (ST) in rats of different ages (78). SN has the greater content of both compounds: 3-4-fold more than ST of the young animals. During the aging process there is a significant decrease (50 %) of 1MeTIQ concentration in SN. However, 1MeTIQ seems to increase in ST although this change is not significant. The TIQ levels change in the same directions as 1MeTIQ in both structures and ages, but these changes are not statistically significant. Because the ratio 1MeTIQ/TIQ could be important, it was suggested that the protective action of MeTIQ could be related to its possible action against other endogenous compounds with neurotoxic actions, such as TIQ. In fact, one possible cause of the major incidence of PD during aging could be due to a decrease in the levels of protective compounds such as 1MeTIQ which would produce an increased vulnerability to environmental neurotoxins. 1MeTIQ in rats prevents the extensive degeneration of dopaminergic terminals induced by MPP+ injection into the striatum (79). In hepatic mitochondria, 1MeTIQ (5 mM) totally prevents complex I inhibition by 20 mM MPP+. 1MeTIQ could protect the activity of this enzyme, avoiding the interaction between MPP+ and complex I (80). A similar phenomenon between a weak and a strong inhibitor of complex I has been previously described (81). 1MeTIQ has been described as being enzymatically formed in the brain by the 1MeTIQ synthesizing enzyme (1MeTIQse) (82). The activity is spread throughout the brain, the highest activity being in the dopaminergic areas (striatum and substantia nigra) and in the cortex. During aging there is a 1MeTIQse activity reduction (50%) in the areas implicated in the etiology of Parkinson disease (substantia nigra, striatum) and in the cerebral cortex. 3.3. Rotenone Dopaminergic toxicity of the MPTP active metabolite MPP+ is mediated by the DAT through accumulation in DA neurons, where it inhibits mitochondrial complex I activity. Moreover, various endogenous and exogenous heterocyclic molecules, which are structurally related to MPTP/ MPP+, such as isoquinolines and beta-carbolines, have been reported to exhibit similar toxic properties on DA cells, which are conferred by their DAT uptake. Taken together, there is a large body of evidence from morphological, molecular biological and toxicological studies indicating that the DAT might be responsible for the selectivity of DA cell death in PD. Recently, rotenone has given new clues to our current understanding of PD pathogenesis. Rotenone is a classical, high affinity inhibitor of complex I, which has been widely used to know the specific activity of the complex. Unlike MPP+, rotenone is extremely lipophilic, by which it freely crosses the blood brain barrier and biological membranes, thus reaching the brain rapidly (83). 3.3.1. In vivo rotenone models of Parkinson’s disease Strikingly, in vivo rotenone administrations to rodents have mimicked typical histopathological features of PD, including the formation of Lewy bodies. Heikkila and colleagues had already reported that acute stereotaxic injection of rotenone into the rat median forebrain bundle induced striatal DA depletion (84). Subsequent studies administered rotenone orally (85) and subcutaneously (86) without selective damage to the nigrostriatal dopaminergic system. Ferrante et al (87) demonstrated that a high-dose intravenous infusion of rotenone for 7 9 days in rats resulted in selective tissue damage in the striatum and globus pallidus, but not in the substantia nigra (87). Using minipumps for chronic intravenous rotenone infusion for 735 days, Betarbet and colleagues clearly demonstrated selective pathologic and biochemical changes in the nigrostriatal dopaminergic system (88). In this study, rotenone partially inhibited complex I uniformly throughout the brain accompanied by selective neurodegeneration of the nigrostriatal dopaminergic system. The optimal dose for inducing parkinsonian-like features was determined to be 2-3 mg/kg per day in Lewis rats. A principal limitation of this model is the difficulty associated with jugular vein Mitochondria and neurodegenerative diseases 990 administration of rotenone. Sherer et al (89) described a simpler but efficient way of administering rotenone by means of chronic subcutaneous exposure to 2.0-3.0 mg/kg/day of rotenone with osmotic minipumps for 28 days. According to the authors, chronic subcutaneous exposure to rotenone induced selective nigro-striatal dopaminergic degeneration and formation of alphasynuclein-positive cytoplasmic aggregates with no signs of degeneration in other brain areas (89). Chronic administration of rotenone has also been performed by intraperitoneal injections (1.5-2.5 mg/kg/day for 2 months) in Sprague-Dawley rats (90). In this study, there was a dopamine depletion in striatum and prefrontal cortex along with catalepsy. However, high variability observed among different species and the exact nature of the mechanism by which rotenone induces cell death remain to be established. If behavioural impairment in response to rotenone is due to striatal dopaminergic dennervation, then L-DOPA administration should be able to improve motor deficits. Few reports have addressed this issue. Chronic exposure of Drosophila to sublethal doses of rotenone is accompanied by selective loss of dopaminergic neurons inducing locomotors deficits (91). In this study, L-DOPA administration prevented locomotor deficits but not neuronal degeneration, while melatonin treatment prevented both locomotor deficits and neuron degeneration, thus suggesting that rotenone-induced cell death, at least in Drosophila, results from an oxidative process, and that locomotor deficits are dopamine-dependent. Furthermore, Alam and Schmidt, (90) reported that hypokinetic behaviour, which mimics parkinsonian behavioural symptoms, e.g. akinesia and rigidity (catalepsy) in rats, was reversed by L-DOPA treatment. Similar results were found by Alam et al (92) in response to bilateral injection of 3 microg of rotenone into the medial forebrain bundle. Under these conditions, rotenone induced a 50 % reduction in striatal dopamine levels along with motor deficits. With all this a pair of questions arise in the rotenone model of PD: a) what is the mechanism(s) of the rotenone-induced cell death?; b) what makes the nigrostriatal dopaminergic more susceptible to rotenone neurotoxicity ? 3.3.2. Rotenone and oxidative stress Sherer et al (93,94) investigated potential mechanisms of rotenone toxicity. In the first study (93), they used a chronic in vitro model based on treating neuroblastoma at low concentrations of rotenone with the aim of partially inhibiting complex I activity to mimic that seen in PD. They found that oxidative damage correlated temporally with alpha-synuclein aggregation and importantly, chronic rotenone treatment sensitized cells to further oxidative challenge. This study would highlight the role of the rotenone-induced oxidative damage to further induce cell death. In keeping with this view, in a different study, Sherer et al (94) concluded that rotenone toxicity did not result solely from a bioenergetic defect but rather from oxidative damage. Firstly, in neuroblastoma cells, rotenone caused dose-dependent oxidative damage, and rotenone toxicity was prevented by two antioxidants, alphatocopherol and coenzyme Q10 (61). Secondly, using a chronic midbrain slice model, rotenone-induced oxidative damage and toxicity to mature dopaminergic neurons were also attenuated by antioxidant treatment (61). Thirdly, rotenone infusion caused relatively selective oxidative damage to certain dopamine-rich brain regions known to degenerate in PD, thus suggesting that free radicals originated by rotenone and dopamine metabolism synergistically interact to induce cell death (94). In addition, micromolar Ca2+ concentrations in the presence of rotenone strongly increase the detection of ROS production in isolated rat forebrain mitochondria (95). Ca2+-stimulated mitochondrial ROS release could this participate as an important link between the partial mitochondrial complex I inhibition and oxidative damage observed in PD. Sipos et al (96) reported that a moderate level of complex I inhibition by rotenone leads to significant ROS formation. Since a partial inhibition of complex I is obtained in response to chronic in vivo administration of rotenone, the finding by Sipos et al (96) would justify the intense oxidative stress in the rotenone model of PD. A low dose of rotenone at 1.5 mg kg−1 in Sprague–Dawley rats enhanced NO generation in different brain areas including striatum and frontal cortex along with lipid peroxidation-like products (97), thus suggesting that NO-derived products may participate in the toxicity of rotenone. In keeping with this view, He et al (86) found that following the low dose of rotenone (1.5 mg kg-1 i.p.) there is a significant increase of neuronal NOS activity along with increased generation of peroxynitrite in the striatum, which may explain the possible involvement of NO in the induction of selective nigro-striatal dopaminergic damage by rotenone. In fact, administration of the neuronal NOS inhibitor 7-nitroindazole significantly attenuated the increased NOS activity and 3-NT production, and provided significant protection against rotenone-induced nigrostriatal injury (98). An endoplasmic reticulum oxidative stress response to rotenone which may induce cell death was suggested (99). Accumulation of damaged oxidized proteins could interfere with the cellular protein degradation machinery, thereby causing the ER to retain unfolded proteins (99). Although it is clear that the main mechanism of rotenone-induced cell death is associated with inhibition of complex I, it should be considered the potential role of activated microglia. Reactive microglias are seen in the basal ganglia following rotenone treatment, even before development of nigrostriatal dopaminergic lesions (100,101). Gao et al (98,100) observed the deleterious role of microglial NADPH oxidase in the rotenone model of PD. These studies were conducted in primary neuronenriched and neuron/glia cultures from rat mesencephalon (102) or NADPH oxidase—null (gp91phox-/-) mice (100). It is yet to be established if a similar effect is seen in adult animals under in vivo conditions. NADPH oxidase generates O2 - towards the extracellular space, the effect being toxic to DA neuron. In addition, the increase of intracellular ROS in microglia is critical in the Mitochondria and neurodegenerative diseases 991 proinflammatory signalling in professional phagocytes and serves to amplify the production of neurotoxic proinflammatory factors, such as TNF-alpha (103). Lipopolysaccharide (LPS) is the active immunostimulant of the cell wall of Gram-negative bacteria that is responsible for triggering the cascade of events following bacterial infection (104,105). A single intranigral injection of LPS induces loss of nigral tyrosine hydroxylase (TH)-immunopositive neurons with concomitant decreases of DA and its metabolites in the nigrostriatal system (106). Interestingly, the SN is especially susceptible to LPS-induced neurotoxicity as compared with other brain areas (107). The neurotoxic effect of LPS has been observed in mesencephalic cell cultures (108-110). Gao et al (111) tested the possibility that inflammation and inhibition of complex I may act synergistically to further induce cell death. They used primary mesencephalic dopaminergic cultures that were exposed to non-toxic doses of rotenone and the inflammogen LPS and found that they acted synergistically to induce dopaminergic degeneration (111). NADPH oxidase-mediated production of O2 - in rotenone and LPS- activated microglia appears as an important mediator of the synergistic neurotoxicity (111). 3.3.3. Rotenone and the proteasome. Two major features of parkinsonian brains are decreased complex I activity and reduced activity of the ubiquitin proteasomal system (UPS). Formation of proteinaceous inclusions is a well-established feature in the rotenone model of PD (88,112). Microinfusion of low doses of the proteasome inhibitors, lactacystin and epoxymycin, in the striatum resulted in the loss of striatal TH –immunoreactive terminals along with retrograde degeneration of substantia nigra dopaminergic neurons and formation of neuronal inclusions reactive to alphasynuclein (113). With these precedents, Hoglinger et al. (114) tested the possibility that inhibiton of complex I and reduced activity of the UPS could be related events. Interestingly, the authors found that reduced activity of the UPS increases the vulnerability of mesencephalic neurones in vitro to rotenone and that complex I inhibition can decrease proteasomal activity, suggesting a possible mechanism by which the UPS might be impaired in sporadic PD. Further evidence in favour of a relationship between inhibition of complex I activity and dysfunction of the UPS was obtained by Shamoto-Nagai et al. (115) that observed that rotenone greatly inhibited the UPS in neuroblastoma SH-SY5Y cells The reduction in activity was neither due to ATP depletion nor to reduction of the protein level, suggesting that accumulation of oxidized proteins in response to rotenone treatment may underlie the subsequent inhibition of the proteasome (115). Finally, Betarbet et al. (116) examined the 20S proteasomal activity in rotenone-infused rats, and found a significant reduction in ventral midbrain regions. 3.3.4. Rotenone and apoptosis Ahmadi et al (117) reported that low concentrations of rotenone induce caspase-dependent apoptosis in mesencephalic dopaminergic neurons, a process which was significantly inhibited by the caspase-3 inhibitor, DEVD. Rotenone has been shown to induce apoptosis in human dopaminergic SH-SY5Y cells, a process that was demonstrated to be mitochondrial caspasedependent (118-121). Caspase-12 is an endoplasmic reticulum-specific caspase which is known to participate in endoplasmic reticulum stress-induced apoptosis. Kitamura et al. (118) found that rotenone induced the degradation of procaspases-12 before DNA fragmentation and cell death, thus suggesting that endoplasmic reticulum stress is also involved in the rotenone-induced apoptosis. As pointed out earlier, Ryu et al. (99) suggested that oxidative stress caused by rotenone may be responsible for inducing endoplasmic reticulum stress. In keeping with this view, formation of ROS has been shown to precede apoptotic events such as caspase-3 activation and poly(ADP-ribose) polymerase cleavage in SH-SY5Y cells (120). Different studies have attempted to establish the apoptotic pathways triggered by rotenone, especially in SH-SY5Y cells. The apoptotic death of dopaminergic cells was preceded by the activation of NF-kappaB (122). p38 and JNK but not ERK 1/2 signalling pathways were reported to participate in the caspase 3 activation in response to rotenone (121). In addition, rotenone was reported to induce Bad dephosphorylation, a pro-apoptotic member of the bcl-2 family, which precedes further activation of caspase 9 and apoptosis (123). Since apoptosis is a remarkable feature of dopaminergic cells in response to rotenone, different studies tested the potential antiapoptotic properties of different compounds. Thus, treatment of SH-SY5Y cells with different antioxidants including myricetin, fraxetin and N-acetylcysteine prevented the rotenone-induced caspase 3 activation and cell death (120). Cyclopentenone prostaglandin A1, an inducer of heat shock proteins, dramatically increased cell survival, attenuating both the apoptotic and the necrotic cell deaths of SH-SY5Y in response to rotenone. Search for compounds with neuroprotective properties are encouraging with the aim of finding potential neuroprotective pharmacological strategies to treat Parkinson´s disease. Activation of KATP channels in PC12 cells has been shown to confer protection against mitochondrial complex-I inhibition-induced cell death (124,125). However, it awaits further elucidation of the signal transduction elements up- and downstream of mitochondrial KATP channels. 3.3.5. Rotenone and the dopaminergic system The higher toxicity of rotenone towards the dopaminergic neurons is still under investigation, taking into account that some data suggest more generalized damage. Lapointe et al. (126) subjected male Lewis rats to systemic treatment of rotenone via subcutaneous delivery at a dose of 2.5 mg/kg/day and found that 75% of the animals showed signs of illness that required sacrifice. Significantly, autopsy analysis demonstrated peripheral damage to many organs that might be related to hypokinesia seen in different reports. Another major issue dealing with the rotenone model of PD is the high rate of Mitochondria and neurodegenerative diseases 992 lethality in response to low doses of rotenone ranging between 2-2.5 mg/kg/day. In the study by Lapointe et al (126), less than 20% of all rotenone-treated animals showed loss of striatal dopaminergic denervation. Similar results were found by Fleming et al (127). Hoglinger et al (112) investigated the differential vulnerability of various brain structures to generalized complex I inhibition induced by intravenously infusion with rotenone (2.5 mg/kg/day) for 28 days in male Lewis rats. The authors found a pronounced loss of spontaneous locomotor activity that did not correlate with a moderate loss of only 55% of striatal dopaminergic fibres, thus suggesting that factors other than dopaminergic denervation may be involved in the behavioural changes induced by rotenone. In keeping with this view, the study of Hoglinger et al (112) also found degeneration of non-dopaminergic systems, including striatal dopaminoceptive DARPP-32-immnoreactive projection neurones (dopamine- and cAMP-regulated phosphoprotein) and cholinergic interneurones. In addition, the study of Hoglinger et al (112) found degeneration not only in basal ganglia but in different brain stem nuclei as well, concluding that substantia nigra is not more susceptible to generalized inhibition of complex I than other basal ganglia or brain stem nuclei. However, a specific vulnerability of the dopaminergic system to the inhibition of complex I seems to be unquestionable. Likely the more diffuse damages could be related to the variability observed among different cell types along with different ways of administration and used concentrations. The highest susceptibility of dopaminergic neurons to rotenone has been described in different types of cells. In culture, rotenone killed more immortalized midbrain dopaminergic differentiated (MN9D) cells than non-dopaminergic differentiated (MN9X) cells (128). Oxidative stress played an important role in rotenoneinduced neurodegeneration of MN9X cells. In contrast, disruption of energy supply played a more important role in MN9D cells: rotenone decreased mitochondrial membrane potential and ATP levels in MN9D cells more than in MN9X cells (128). In the study by Betarbet et al. (88), rotenone (2-3 mg/kg per day in Lewis rats) partially inhibited complex I uniformly throughout the brain accompanied by selective neurodegeneration of the nigrostriatal dopaminergic system. However, the question that arises deals with the primary target of rotenone action; i.e. the cell body region in substantia nigra or the nerve ending region in striatum. Zhu et al. (129) have tested the effect of chronic (3 weeks) subcutaneous rotenone infusion (2 mg/kg) on the integrity of the nigrostriatal system in adult male Lewis rats and found that only half of the animals treated with rotenone presented a diffuse reduction in striatal TH-IR staining without significant loss of nigral DA neurons, thus suggesting an earlier effect of rotenone on DA nerve terminals rather than on DA cell bodies. This study would suggest that the striatum is the primary target of rotenone that would precede a subsequent retrograde degeneration of the DA neurons, as initially suggested by Betarbet et al. (88). In keeping with this view, longer exposure (4 weeks) has been shown to induce both a diffuse reduction in striatal TH-IR and a significant loss of DA cell bodies in the substantia nigra (89,112). The endogenous levels of dopamine have been suggested to contribute to dopaminergic cells selective vulnerability. Sakka et al. (130) reported that treatment of mesencephalic cultures with alpha-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, protected dopaminergic neurons against the chronic treatment with lowconcentration of rotenone. Further evidence that dopamine itself contributes to rotenone toxicity is that inhibition of dopamine metabolism significantly reduced rotenone toxicity (131). Maragos et al. (132) studied the effect of mitochondrial inhibitors on the striatal DA uptake activity. They found that in spite of all of the toxins inhibiting (3H)DA uptake, there was a large variation in their inhibitory potencies, the rank order being rotenone >> cyanide > azide > 3-NPA >> malonate. This suggests that the mechanism by which extracellular DA is increased by mitocondrial toxins involves factor other than mitocondrial ATP production or oxidative stress. A similar study was carried out by Marey-Semper et al. (133) that compared the inhibitory effects of rotenone on the uptake of dopamine, serotonin, noradrenaline and GABA in mouse striatal synaptosomes, and of dopamine, serotonin and GABA in cultured mesencephalic neurons. In both preparations, the uptake of dopamine was much more affected by rotenone than that of other neurotransmitters. Apparently, intrinsic metabolic properties of the nigrostriatal dopaminergic neurons explain the strong inhibition by rotenone of striatal dopamine uptake. We studied the possible constitutive metabolic deficiency that could account, at least in part, for the selective vulnerability of the dopaminergic terminals to the action of rotenone (134) by studying the effect of inhibitors of the respiratory chain (rotenone, antimycin and KCN) on the maximal respiratory rate of synaptosomes and isolated synaptosomal mitochondria from different brain areas, i.e. cortex, hippocampus and striatum, and in isolated liver mitochondria. The results showed no differences in the effect of the inhibitors in isolated mitochondria. In contrast, a greater inhibition was found in striatal synaptosomes than in cortical or hippocampal synaptosomes when rotenone was used. Moreover, nomifensine or GBR-12909, inhibitors of the dopamine uptake system, had a protective effect on rotenone inhibition. This study indicates the great importance of the dopamine uptake system in the vulnerability of the striatal dopaminergic system. We reached similar conclusions when the rotenone effect on the nigrostriatal system was tested by microdialysis (135). A hypothesis, which would justify the selective vulnerability of nigral dopaminergic neurons to the neurotoxic effect of rotenone, relies on the microtubuledepolymerizing activity of rotenone (131). In this study, rotenone-induced microtubule depolymerization on midbrain dopaminergic neurons in culture disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Supporting the view that the microtubule-depolymerizing activity of rotenone may Mitochondria and neurodegenerative diseases 993 Table 1. Inhibitors of complex I Pesticides Natural compounds Benzimidazole Acetogenins from Annonacae plants (custard apple, paw) Bullatacin Antibiotics from Myxobacteria 6-Chloro- benzothiadiazole Piericidins1 from Streptomyces strains Cyhalothrin Rhein from Rhubarb Fenazaquin Rotenoids from Leguminosae plants Fenpyroximate Hoe 110779 Pyridaben Pyrimidifen Sandoz 548A Tebufenpyrad Thiangazole 1 More potent than Rotenone. References: 134,135. be involved in the selectivity of rotenone towards the dopaminergic system, toxicity was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole (131). 3.4.6. Other inhibitors of complex I. Actually there are some pesticides and natural compounds that are inhibitors of complex I and some of them are used in agriculture (136,137) (Table 1 ). Some of these compounds could contaminate food, air, etc. and could be involved in the slow degeneration of the dopaminergic system. Its concentration could be especially related to its selectivity. Moreover, this could also depend on the age of the receptor along with genetic susceptibility. 4. TOXINS THAT PRODUCED A NEURODEGENERATION LIKE THAT FOUND IN HUNTINGTON DISEASE: INHIBITION OF SUCCINATE DEHYDROGENASE Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder characterized by involuntary choreiform movements, along with memory disturbances, psychiatric manifestations, frontal type cognitive abnormalities, decreased verbal fluency, and poor recall of recently learned information (138-141). HD patients show progressive cell loss in brain regions belonging to the basal ganglia, particularly the striatum (142,143). The genetic mutation associated with HD has been localized in the gene named IT15, on the short arm of chromosome 4, and consists of an enlarged repeat of CAG triplets in the 5' coding region (144). This genetic defect seems to lead to an impairment of oxidative phosphorylation has been proved in vivo with the detection, by nuclear magnetic resonance spectroscopy, of an increased concentration of lactate in the cerebral cortex and in the striatum in HD patients (145). Moreover, several studies have reported decreased striatal glucose metabolism (146-151), and abnormalities in the mitochondrial electron transfer chain (152-155). Ludolph et al. (156) showed that contaminant, toxin-generating fungus Arthrinium spp, was the culprit of encephalopathy and the late onset of dystonia appearing in children after the ingestion of sugarcane described in The People’s Republic of China. Children seem especially vulnerable to the toxin which produces sudden-onset nausea, vomiting, abdominal pain, and diarrhea; afterward, they develop double vision, somnolence, nystagmus, convulsions, decerebrate rigidity and coma. Computerized tomography of the disabled children reveals bilateral hypodensity of the putamen and, to a lesser extent, the globus pallidum (157,158). The mycotoxin 3- nitropropionic acid (3-NPA) from Arthrinium has been reported as the cause of mildewed sugarcane intoxication (156). 3-NPA is an irreversible inhibitor of the Krebs’ cycle enzyme succinate dehydrogenase (SDH), localized in the inner mitochondrial membrane and responsible for the oxidation of succinate to fumarate (159,160). The 3-NPA inhibits Complex II of the respiratory chain as well as Krebs’ cycle (159). Animals chronically treated with 3-NPA develop a HD-like syndrome. Chronic systemic administration of low doses of 3-NPA produces excitotoxic-like lesions regionally restricted to striatum in rats (161-164) and nonhuman primates (165,166). Histologically, these 3-NPA lesions are characterized by marked loss of medium-sized spiny neurons, astrogliosis, dopaminergic axon sparing, preservation of nicotinamide adenine nucleotide phosphate (NADPH)-diaphorase positive striatal interneurons and alterations in the dendritic morphology of medium spiny neurons (163,167). These changes are strikingly similar to those described in HD striatum (168-170). Moreover, animals with 3-NPA lesions also develop behavioral characteristics of HD. Rats chronically treated with low concentration of 3-NPA showed spontaneous motor symptoms including mild dystonia, bradikinesia and gait abnormalities. In these animals, the degree of striatal neuronal loss was significantly correlated to the severity of spontaneous motor abnormalities, as is the case for HD (171). In addition, Palfi et al. (166) showed that chronic 3- NPA treatment in primates replicates the basic histological and pathophysiological triad of HD, including frontostriatal syndrome of cognitive impairment. 4.1. Pathways of cells death induced by 3-NPA The most generally accepted hypothesis for the neuronal cell death induced by 3-NPA has, at least, three different components: a) depletion of ATP levels, produced by a deficit in energy metabolism as a consequence of Complex II inhibition; b) excitotoxicity produced by energy impairment and membrane depolarization along with NMDA toxicity via the release of a voltage-dependent Mg2+ block, leading to the influx of Ca2+ and subsequent activation of a “cell-death” pathways (167,172-175), and c) oxidative stress produced by the formation of reactive oxygen and nitrogen species, also as a consequence of impaired energy metabolism (176). 4.1.1. Energy depletion SDH activity was reduced by 75% in whole brain mitochondrial suspensions by the injection of lethal doses Mitochondria and neurodegenerative diseases 994 of 3-NPA (177). Similarly, SDH activity in brain showed a profound decrease 30 min after the injection of sublethal doses of 3-NPA, shown by qualitative SDH histochemistry. Brouillet et al. (178) demonstrated that the threshold of irreversible inhibition by 3-NPA in vivo in the brain to produce neurotoxicity was 50-60% of the control levels. In addition, Pang and Geddes, (179), also described that ATP depletion was an early event in the 3-NPA-induced neuronal degeneration, and that decline in ATP was exacerbated by glutamate in primary cultures of rat hippocampal neurons. Erecinska and Nelson (180), using synaptosomal preparations, described the time course of events produced by 3-NPA toxicity. First of all, there was a decrease in the phosphocreatine/creatine ratio along with an increase in the lactate/pyruvate ratio. Afterward, severe decreases in ATP/ADP and GTP/GDP ratios were produced. Moreover, the administration of either creatine or cyclocreatine leads to increase in the brain concentrations of phosphocreatine and phosphocyclocreatine respectively, with a significant protection against malonate (MA) and 3-NPA neurotoxicity without depletion of ATP (181). 4.1.2. Excitotoxicity It is well accepted that impairment in energy metabolism, that depletes ATP, causes the failure of ATP- dependent ion pumps and channels which results in depolarization of the cell (182). As glutamaceptive neurons depolarize, the Mg2+ block in the NMDA receptor channel is released, thus allowing even physiological concentrations of glutamate to activate the receptor channel (163). This activation of NMDA receptors resulting from partial energy impairment could also be, at least partially, independent of the effect of the Mg2+ block (175). Activation of the NMDA receptor channel leads to Ca2+ influx (183), which cannot be reversed due to the lack of ATP to run the ion antiporter pump. Thus, intracellular Ca2+ overload leads to neurotoxicity. Consequently, compromised energy metabolism leads to secondary excitotoxicity and neurodegeneration in glutamate target regions (184), since decortication removing the cortico-striatal glutamatergic input attenuates the lesions (161,162). In addition, cell loss can be prevented by pretreatment with NMDA receptor antagonists. Significant prevention of the neuronal death induced by 3-NPA toxicity in vitro and in vivo, in spite of the ATP concentration continuing to be severely depleted, has been described (185). Blockade of excitatory transmission by NMDA glutamate receptor antagonists such as MK801 largely prevents striatal damage due to systemic 3-NPA or intrastriatal MA (161,186,187). This shows that the selective blockade of the NMDA receptor is able to reduce neuronal death induced by 3-NPA toxicity (185). 4.1.3. Oxidative stress This stress is due to the actions of reactive oxygen species (ROS) such as O2 - and H2O2 produced in excess by the mitochondrial respiratory chain. Secondary activation of Ca2+-dependent enzymes such as phospholipase A2 (production of inflammatory mediators) and nitric oxide synthase (production of the second messenger, NO) also promotes the production of ROS (188,189). Another risk factor is the excessive release of dopamine, which generates H2O2 when metabolized (190). In vivo lesioning by 3-NPA or MA causes increases of various parameters of oxidative stress, such as 3-nitrotyrosine, 8-hydroxy-2-deoxyguanosine and malondialdehyde (16,191,192). Moreover, 3-NPA induced in vivo protein oxidation in striatal and cortical synaptosomes (193). Schulz et al. (194) showed that the systemic administration of 3-NPA increased the production of hydroxyl radicals (HO.) in striatum measured by the salicylate assay. Free radicals scavengers, free radical spin trap DMPO and nitric oxide synthase inhibitors significantly attenuated both MA and 3-NPA neurotoxicity (192,194). The selective lesions in the basal ganglia produced by 3-NPA were attenuated in mice, overexpressing Cu,Zn-SOD (193) and in Bcl-2 overexpressing mice, in which the conversion of 4- hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (as a measure of free radical production) was completely blocked (196). In addition, Calkins et al. (197), pointed out the implication of nuclear factor erythroid 2-related factor 2, involved in the antioxidant response element, as an essential inducible factor in the protection against complex II inhibitor-mediated neurotoxicity. 4.2. Specificity of the 3-NPA toxicity There are two different kinds of specificity: the brain structure and the kind of cell mainly affected. The striatum is the most affected brain region (161,162,198- 200), and the GABAergic neurons, which make up the bulk of the neurons in the striatum, the cells which are preferentially vulnerable to 3-NPA toxicity, with relative sparing of NADPH-diaphorase and cholinergic neurons (171,199). Glial mitocohondria are morphologically well preserved after 3-NPA toxicity (201), and the selective inhibition of tricarboxylic acid cycle of GABAergic neurons after 3-NPA was seen in vivo, while the tricarboxylic acid cycle of glial cells remained uninhibited (197). Astrocytes or endothelial cells may be the first to fall, which could be involved in 3-NPA-induced bloodbrain barrier disruption (179,202,203). The excitotoxic nature of these lesions could explain the specific vulnerability of GABAergic neurons and the relative sparing of cholinergic and NADPH- diaphorase positive interneurons. In addition, their deleterious effects could be prevented by prior administration of NMDA-receptor antagonists or removal of glutamatergic striatal afferents. Furthermore, subtoxic doses of the mitochondrial inhibitor malonate, has been shown to potentiate the excitoxicity of NMDA (204). Both striatum and hippocampus receive glutamatergic inputs which predisposes them to excitotoxic mechanisms. In addition, SDH inhibitors produce long-term potentiation of the NMDA-mediated corticostriatal excitation in spiny neurons, but not in cholinergic interneurons (205). Saulle et al. (206) have demonstrated that inhibition of mitochondrial complex II produces opposite membrane potential changes in striatal spiny neurons and in cholinergic interneurons. 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Rose: Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity. Proc Natl Acad Sci U S A 93, 1330-4 (1996) Key Words: Parkinson´s Disease, Huntington´s disease, Rotenone, 3-NPA, isoquinolines, Review Send correspondence to: Professor Alberto Machado, Departamento de Bioquimica, Bromatologia, Toxicologia y Medicina Legal, Facultad de Farmacia, c/ Profesor García González 2. 41012-Sevilla, Spain. Tel. and Fax: 34- 9546752, E-mail: [email protected] http://www.bioscience.org/current/vol12.htm