Mechanisms of MPTP Neurotoxicity Causative of The Production

[visionarybear]

hey guys,
jus thought id post a paper i recently had to write for uni, might be of some interest to someone..
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Mechanisms of MPTP Neurotoxicity Causative of The Production of Parkinsonian Syndromes

Parkinson’s Disease (PD) is a degenerative movement disorder. PD is characterized clinically by resting tremor, bradykinesia, postural instability and rigidity. Post mortem examinations of PD patients also show a selective decline in dopamine (DA) neurons of the substantia nigra pars compacta (SNpc) in preference to DA neurons in the ventral tegmental area (VTA) and smaller decreases in other monoamine neuron dense areas such as the locus coeruleus, intracellular proteinaceous inclusions termed lewy bodys (LBs) and extracellular α-synuclein containing aggregates (reviewed by Dauer and Przedborski, 2003). Clinically, symptoms of classical PD is termed parkinsonism until post mortem conformation of molecular patho-physiology. The remainder of this essay will focus on the induction of parkinsonism by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolites.
   MPTP is a bi-product of the illicit synthesis of 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP) under altered reaction conditions (Langston et al., 1983). MPTP and MPPP are meperidine analogs. Severe non-resolving parkinsonism was observed in 4 Californian drug users by Langston et al. in 1983. The drug users had injected intravenously the MPTP containing drug obtained under the guise of ‘synthetic heroin’ for approximately 1 week before onset of symptoms caused hospitalisation (Langston et al., 1983). Parkinsonism as result of MPTP intoxication shows all but 2 of the hallmark features of classical parkinsonism (Langston et al., 1983). MPTP selectively destroys DA neurons in the SNpc but leaves other monoamine rich areas intact. LBs have not been observed in any cases of MPTP intoxication in humans but, as only 4 cases have come to autopsy, it is far from conclusive (Langston et al., 1999). It is hypothesised that this may be due to the acute nature of MPTP intoxication, the degeneration being too sudden for LBs to form (Betarbet et al., 2000). MPTP has become the gold standard in animal models for the novel treatment of PD.
   MPTP is a highly lipophilic molecule that absorbs easily across the gut and blood brain barrier (BBB) (reviewed by Dauer and Przedborski, 2003). MPTP on its own is a non-toxic entity (McKinley et al., 2005; Salach et al., 1984; Singer et al., 1985). It becomes toxic when it is metabolised into 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP+) by mainly mono-amine oxidase type b (MAOB) and is further oxidised to the 1-methyl-4-phenylpyridinium ion (MPP+) by MAOB and by spontaneous oxidation (Salach et al., 1984; Singer et al., 1985). This is supported by the data found by Langston et al. (1984) that pargyline, a MAO inhibitor, prevented MPTP toxicity in squirrel monkeys (Saimiri sciureus). MPTP was administered by four 2mg/kg intraperitoneal injections 2 hours apart in a single day to all monkeys. Two 2 of the 3 groups also received pargyline by one of 2 dose regimes. 50mg/kg intraperitoneal (IP) injection 30min prior to MPTP administration or oral 5mg/kg/day for 4 days prior and 5mg oral 1hour before each MPTP injection. Histological examination and assays for MPTP and MPP+ were preformed. It was found that pargyline protected against chronic nerve damage with no visible neuronal loss in the areas examined. The assays revealed that blockage of MAO enzymes caused approximately a 10 fold decrease in MPP+ level in cortical areas examined. This indicates the importance of MAO in MPP+ production. More specifically, McKinley et al. (2005) showed that l-deprenyl (MAO-B inhibitor) protected against MTP induced neuronal damage in zebra fish, implicating MAO-B as the likely isoform responsible. The transport of MPP+ into the extracellular space is by an unknown mechanism but is presumed to be via a transporter protein as the charged species would be unable to diffuse across the lipid membrane unaided (reviewed by Dauer and Przedborski, 2003).  
   MPP+ has a high affinity for several mono-amine transporters including those of serotinergic, adrenergic (NET) and dopaminergic (DAT) neurons (reviewed by Dauer and Przedborski, 2003). DAT has been shown to be directly linked to sequestration of MPP+ by dopamine neurons, facilitating subsequent damage and neuronal loss (reviewed by Dauer and Przedborski, 2003; McKinley et al., 2005). McKinley et al. demonstrated the protective effect seen in DAT knockdown zebra fish. Zebra fish embryos injected with DAT morpholino (4.8 ng), or saline vehicle, and later incubated with 5 or 10 μ g/mL MPTP solutions or vehicle for 3days. The amount of MPTP damage was measure by morpholocial studies and by behavioural tests. DAT knock down fish were more responsive than MPTP only fish, showing a response to touch but not swimming away, as seen in the non MPTP treated fish. MPTP treated wild type fish were unresponsive to touch and completely immobile. Morphological quantification of the relative neuronal loss was measured as % neuronal area compared to untreated wild type embryos. The results paralleled the observed behaviors. These results emphasize the importance of DAT in MPTP induced damage.
   DAT on its own does not provided a reliable measure of sensitivity to MPP+ damage. Once MPP+ has entered DA neurons, it may be taken up into synaptosomal vesicles by vesicular monoamine transporter 2 (VMAT2) (Liu et al., 1992).Liu et al. (1992) showed that genetic transformant Chinese hamster ovarian (CHO) fibroblasts can become MPP+ resistant when they acquire VMAT2. This was achieved by transfecting a cDNA expression library from rat phenochromocytoma PC12 cells into CHO fibroblasts and incubating them in various concentrations of 3H MPP+ (up to 1000 μM). Preparations were tested in the presence of 1 μM reserpine, an inhibitor of vesicular transporters, which caused a reversion to the wild-type sensitivity levels. This showed that the resistance was dependent on reserpine-sensitive VMAT2. This means that a better predictor of MPTP sensitivity is the ratio of DAT to VMAT2 in DA neurons. This ratio has been attributed to the selectivity of MPTP for SNpc and not DA neurons of the VTA (reviewed by Dauer and Przedborski, 2003). This also has implications in the selective neuronal loss idiopathic PD (reviewed by Dauer and Przedborski, 2003).
   MPP+ may also stay in the cytosol and react with proteins that bear a negative charge (Klaidman et al., 1993). MPP+ has been shown to have strong binding interactions with neuromealanin (D'Amato et al., 1986). This interaction may act a pool of MPP+, able to dissociate and exert a toxic effect long after the initial exposure to MPTP (D'Amato et al., 1986). MPP+ has also been shown to up regulate alpha-synuclein expression and aggregate with it to form intracellular inclusions (Kalivendi et al., 2004). These aggregations may cause cellular dysfunction and apoptosis by steric hinderance to normal cellular transporters and organelles (Kalivendi et al., 2004).  
   MPP+ is quickly concentrated into the mitichondria from the cytosol in an energy dependent fashion mostly due to the membrane potential (Ramsay & Singer, 1986). Once in the mitochondria, MPP+ is an inhibitor of NADH CoQ1 reductase (complex 1 of the electron transport chain), (Bates et al., 1994; Maharaj et al., 2006). Bates et al. (1994) demonstrated the effects on mitochondrial respiration, ATP generation and radical formation, in isolated brain mitochondria, under the influence of complex 1 inhibitors. 0.5mg isolated mitochondria was incubated with either 10 â€" 100 μM MPP+, 10 μM 4’hepatyl MPP+ or 5 μM rotenone (Positive control). Preparations were incubated for 10min at 30 ºC and various assays conducted to measure O2 consumption in stage 3 and 4 respiration, adenosine triphosphate (ATP) production, complex 1 activity and Radical formation. O2 consumption in stage 3 respiration was effectively halved by 20 μM MPP+ (Approximate IC50) and 10 μM 4’hepatyl MPP+ against control. ATP production decreased by 1/3 for MPP+ (60 μM, approximate IC50) and by 2/3 for 4’hepatyl MPP+ against controls. This shows the inhibitory potential of MPP+ inside the mitochondria for complex 1 and that MPP+ can inhibit mitochondrial ATP production. If ATP production is critically inhibited, there would be a subsequent collapse of the mitochondrial membrane potential due to the inability of ATP-ase ion pumps to function (reviewed by Dauer and Przedborski, 2003). This could also lead to the formation of reactive oxygen species (ROS) due to DA leakage into the cytoplasm as VMAT2 is unable to maintain concentration gradients into synaptosomal vesicles (reviewed by Dauer and Przedborski, 2003).
   Antioxidants have been shown to attenuate ROS produced by MPP+ and complex 1 interaction (Maharaj et al., 2006). Maharaj et al. (2006) demonstrated the attenuating effects of acetaminophen (APAP) and acetylsalicylic acid (ASA) against MPP+ induced neuronal damage. APAP, ASA or APAP + ASA treatment attenuated the decrease in complex 1 activity produced by MPP+. ASA caused a significant increase in complex 1 activity compared to basal level in controls. This shows that ASA competes with MPP+ for its complex 1 binding site. ROS levels were also shown to be decreased compared to MPP+ alone, with ROS formation in the ASA only treatment group showing decreased ROS formation compared to control levels, showing a link between complex 1 inhibition and ROS generation.
   Przedborski, et al. (1992) showed that ROS contribute to the toxic effects of MPP+. Mice transgenic for Cu/Zn super oxide dismutase (Cu/Zn-SOD) were tested alongside non transgenic littermates. Mice were given three injections of 30mg/kg IP MPTP-HCl or vehicle control 24hrs apart. After 5 days, mice were sacrificed and the brain matter was analyzed. Levels of DA and its metabolites were used as a marker for MPTP toxicity. There was a 50% decrease in DA and its metabolites in non-transgenic mice compared to saline vehicle controls and no significant change in DA metabolite levels in transgenic mice. To determine if this protective effect was due to changes in the brain other than increases ROS scavenging by increases SOD, MPTP penetration across the BBB, MAOB activity, MPP+ sequestration into mitochondria and complex 1 inhibition were measured. There were no significant changes in the brain structure or MPTP metabolism compared to non transgenic littermates. Protection was thus due to increased scavenging of ROS only. ROS were presumably a product of inhibition of complex 1 by MPP+.
   Changes in energy metabolism and ROS formation peak within hours of MPTP administration, long before most neuronal death occurs (Jackson-Lewis et al., 1995). This means it is likely that these immediate effects lead to a down stream effect that ultimately kills the neurons possibly via apoptotic pathways (reviewed by Dauer and Przedborski, 2003). Mitochondrial release of cytochrome c, a mediator of programmed cell death (PCD), and activation of caspase 3 and 9 have been observed in tandem with MPP+ sequestration into the mitochondria (Viswanath et al., 2001). Teng et al. (2006) found that nucling deficient mice were resistant to MPTP toxicity. Nucling is an apoptosis related protein (Teng, 2006). Both wild type and nucling deficient mice were given four IP 15mg/kg MPTP injections 2 hours apart. At 48hrs post treatment, nucling deficient mice showed no signs of MPTP toxicity compared to wild type mice. Nucling deficient mice showed no loss of DA neurons after treatment with MPTP and were negative for apoptosis (via TUNNEL assays) compared to wild type mice. This study demonstrated that apoptotic pathways may be the cause of neuronal death.
   The studies of the complex cascade of events that contribute to the neuronal death endpoint have provided an invaluable research tool which has lead to much advancement in the treatment of PD (reviewed by Dauer and Przedborski, 2003). Two such advancements have been the discovery of the therapeutic effect of deep brain stimulation in reduction of symptoms in advanced PD (Limousin et al., 1998) and the therapeutic benefits of  glial derived neurotrophic factor (GDNF) leading to some functional recovery to previously MPTP lesioned monkeys, aswell as prevention of further degeneration (Kordower et al., 2000).















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