Degenerative mechanism in dopaminergic neurons

1 INTRODUCTION Parkinson’s disease (PD) is a widely occurring neurodegenerative disease that was first described by James Parkinson (Parkinson, 1817) The main clinical symptoms displayed by PD patients include bradykinesis (difficulty in initiating and carrying out movements), tremor, muscle rigidity and jerky movements The more common late-onset, sporadic form of PD affects approximately 2% of the world’s population over the age of 65 years (Hughes et al., 1993) The young are not spared from this disease although the earlyonset familial forms are rare The clinical symptoms were later found to be due to the selective degeneration of neurons containing dopamine as their neurotransmitter within the substantia nigra pars compacta (SNpc), a midbrain structure These cells form part of an interconnecting neuronal circuitry within the brain, which functions to control voluntary motor movement With the progressive loss of up to 80% of these dopaminergic neurons, this circuitry is adversely affected, resulting in the manifestation of the clinical symptoms Given the wide spread nature of this disease, epidemiological studies revealed that the economical burden of PD amounts to between US$7.1b to US$24.5b in USA alone (Siderwof, 2001) Much more are lost indirectly through the decrease in productivity and informal care by family members The cause of PD is unknown No therapeutic agents present are able to cure or delay the onset of PD The best treatment to date is the use of LDOPA, a dopamine precursor L-DOPA is taken orally and it serves to replace the dopamine lost in the SNpc due to the death of the dopaminergic cells (Ball, 2001) However, this treatment could only to reduce the symptoms associated with PD, and is not exactly a long-term cure for it Chronic administration of L-DOPA was recently shown to cause motor fluctuations as well as neuropsychiatric problems such as cognitive impairment (Riley and Lang, 1993) As such, the search for better therapeutic treatment for the cure of PD continues To achieve this, it is imperative to first understand the cause of PD and the basis of the selective loss of dopaminergic cells in the brain Until further studies are done to affirm the molecular pathways of neuronal cell death, it will not be possible to design appropriate and effective therapeutic agents to rescue this disease 1.1 A Short Review on Nigral Degeneration – The Vulnerability of Dopaminergic Neurons Although the etiology of the selective degeneration of dopaminergic cells remained elusive, constant efforts are being made to unravel the phenomena underlying the degeneration process Studies have thus been done on dopaminergic neurons alone, most trying to understand why; of all types of neurons, only the dopaminergic neurons are affected in PD Many factors hence surfaced The utilization of dopamine as the neurotransmitter by these cells is in fact an intrinsic stress factor to the cells (Cohen et al., 1997) Dopamine is metabolized by the enzyme monoamine oxidase (MAO) This is a natural metabolic process to clear the intracellular dopamine within the cytosol However, it was found to generate a significant amount of hydrogen peroxide (H202) The H202 can be further broken down to generate glutathione disulfide (GSSG) Both metabolic products are potentially damaging GSSG reacts spontaneously with thiol groups in proteins to form protein mixed disulfides This reaction compromises the functions of the affected proteins In addition, increased iron level has been noted in the brains of PD patients (Dexter et al., 1989) The H202 generated could react with the iron to generate toxic hydroxyl radicals, which are responsible for damages to membrane proteins via lipid peroxidation Mitochondrial membranes could thus be greatly affected, which in turns, affect its functions The location of MAO on the outer membrane of the mitochondria could thus potentially evoke changes, whether directly or indirectly, at the distant inner membrane These processes have the potential to affect several enzymes located within the mitochondria ATPase, an important enzyme responsible for the generation of cellular energy ATP, is one of them A further postulation is that disturbance to the mitochondria function could lead to the impairment of proton pumping, resulting in a decrease in the mitochondrial membrane potential This would in turn release cytochrome-c, leading to the onset of apoptosis (Desagher and Martinou, 2000) Parkinsonian brains have also been shown to possess decrease level of glutathione (GSH) (Riederer et al., 1989) GSH is an anti-oxidant, and may be involved in the detoxification of H202 generated from dopamine turnover A decrease in the level of GSH would mean a higher basal level of H202 within the cells, thus leading to damages of cellular macromolecules and their subsequent peroxidation The predisposition of dopaminergic neurons to oxidative damages does not stop here The proximity of the mitochondria to these reactive oxygen species (ROS) increases the mutation rate of the mitochondria DNA by 10-20 times (Ozawa et al., 1997) The lack of protective histone-like proteins (Clayton et al., 1974) and the poor DNA repair system in the mitochondria (Shadel and Clayton, 1997) could evoke additive damaging effect to the mitochondrial genome The mitochondrial genome encodes many proteins of the oxidative phosphorylation system This system is responsible for the production of ATP A decline in ATP production due to damages to the mitochondria would result in cellular degeneration, whether it is by apoptosis or necrosis All in all, the above factors have been determined and could act synergistically to produce deleterious effect in dopaminergic cells This increase susceptibility to either environmental or endogenous agents could help explain the selective degeneration of dopaminergic neurons in PD Again, although these facts are known, the exact degenerative mechanisms involved remained enigmatic Thus, efforts are still needed to unravel the mysterious mode of cell death underlying the selective nigral degeneration 1.2 Concluding Remarks on PD Research Review After many years of research, the degenerative mechanisms underlying the selective death of dopaminergic neurons remained a mystery Up till now, there are still no conclusive evidences that state the exact mechanism of death in these cells However, many models have been constantly generated to provide more clues to the understanding of PD With the progressive refinement of such models and the use of more powerful research techniques, the etiology of PD should one day come to light The focus now for scientists working on PD should be to continue in their area of research, thus contributing to the pool of information with their expertise This is thus the aim of the present investigation With the collective efforts of scientists all around the world, the understanding of PD should be within reach, hence giving the many sufferers of PD hope in returning to a normal lifestyle they once had 1.3 The Use of Neurotoxins in PD Research In an effort to understand the etiology of PD, the ideal scenario is of course to extract information directly from patients suffering from PD However, due to the inaccessibility of the mid-brain section and the lack of non-evasive diagnostic tools to study the brain, it has been difficult to obtain valuable information about the real-time events that are happening in the neurons of these PD patients In addition, ethical concerns about the use of PD patients as subjects for studies add on to the current problem As such, pharmacological agents and neurotoxins have been used to develop experimental models in a wide variety of species in order to avoid the problems faced with live human subjects These neurotoxins must demonstrate their abilities to induce and mimic at least certain clinical characteristics found in PD patients, e.g rigidity or the specific loss of dopaminergic neurons in the SNpc There are currently four neurotoxins which have been established to induce most of the characteristics of PD The choice of the neurotoxins employed in each study is dependent on the aspects in which the studies are approached 1.3.1 The Use of Dopamine in PD Research Given the review presented in section 1.1 regarding the intrinsic stress of dopaminerigic cells, it is no surprise that dopamine, the neurotransmitter itself, is used as a neurotoxin to study the disease Dopamine is synthesized and contained in vesicles within the neurons These vesicles serve to regulate the concentration of the neurotransmitter in the cytoplasm and the synaptic cleft (Jonsson, 1971) Dopamine is metabolized over time and this metabolism via MAO can potentially lead to the formation of H202 and dihydroxyphenylacetic acid (Maker et al, 1981) The presence of H2O2 leads to oxidative stress Oxidative stress occurs when the level of reactive oxygen species (ROS) such as H2O2 and hydroxyl radicals is beyond the threshold of what the cells can handle Increased level of ROS is harmful to the cells because it can cause severe damages to DNA, proteins and lipids in the membranes The reaction of the H2O2 with the high levels of iron found in the SNpc region produces hydroxyl radical, which can then immediately react with lipids, DNA, and susceptible amino acids in proteins, resulting in cellular damage (Halliwell, 1992) In addition, the catechol ring of the dopamine molecule can also undergo oxidation spontaneously in the presence of transition metals like iron or enzymatically, to form DA quinone and more ROS (Hastings, 1995) The oxidative stress effects of dopamine can be attenuated by antioxidants such as GSH (Gabbay et al., 1996) Thus, dopamine metabolism can lead to the generation of high levels of ROS within the dopaminergic neurons, thereby leading to oxidative stress and cellular damage Therefore, the exposure of dopaminergic neurons to its own neurotransmitter, even at physiological concentrations (0.1 – 1mM) can induce oxidative stress and subsequent cell death (Ziv et al., 1994) The mode of cell death induced by dopamine seems to indicate towards apoptosis Apoptosis is a regulated mode of cell suicide, the details of which will be discussed in section 1.4 The exposure of both neuronal and non-neuronal cells to dopamine induces several morphological and biochemical hallmarks of apoptosis (Ziv et al., 1994; Stokes et al., 2000) DNA damage, a common downstream effect of oxidative stress was observed with the increased level of p53, a molecule involved in this event (Daily et al., 1999) Bax, a protein also involved in apoptosis, was shown to be activated and the overexpression of its inhibitor Bcl-2, was able to block the dopamine-induced apoptosis (Offen et al., 1997) Although there are clues to the mechanisms in which dopamine causes cell death, the event is not completely elucidated Moreover, the role of dopamine as a neurotoxin to study PD is plagued by the belief that the death mechanisms observed after exposing cells or animals to exogenous dopamine at high concentrations is not representative of the true events happening within the brain of PD patients It has been argued that the main factors for concern should be the intracellular sources of dopamine, as well as its redistribution within the cells Although this is in part true, the relevance of dopamine and its metabolism to the pathology of PD should not be underestimated Studies using dopamine should continue in order to further understand its effects on the intrinsic environment within and around these dopaminergic neurons 1.3.2 The Use of 6-hydroxydopamine (6-OHDA) in PD Research 6-OHDA is a hydroxylated analogue of dopamine and is extensively used as a model to study PD In experimental models of PD, 6-OHDA is directly injected into the striatum, the substantia nigra, or the ascending medial forebrain bundle (for rats) Although 6-OHDA cannot cross the blood-brain barrier, these direct intracerebral injections can reproduce the phenomena of striatal neuronal degeneration, dopamine depletion and the motor impairments that come along with it The relevance of 6-OHDA for PD-related study not only stems from its ability to induce parkinsonism, but also, it has been found to occur naturally in both rat and human brains (Senoh and Witkop, 1959; Curtius et al., 1974) and in the urine of L-DOPA treated PD patients (Andrew et al., 1993) 6-OHDA can be produced by a non-enzymatic reaction between dopamine, H202 and free iron (Linert et al., 1996) Thus, this compound may even potentially play a very important role either in the onset and/or the progress of the disease The use of this compound should yield results relevant to the etiology of PD The presence of 6-OHDA in the striatal region has been suggested to cause nigrostriatal dopaminergic lesions via the generation of ROS e.g H202 (Heikkila and Cohen, 1971) The oxidative stress produced from the high levels of ROS generated in vivo (Kumar et al., 1995) and in vitro (Choi et al., 1999b) can be nullified by the cointroduction of antioxidants (Yamada et al., 1997; Mayo et al., 1999) 6-OHDA was also found to induce biochemical and morphological hallmarks of apoptosis e.g chromatin condensation as detected by TUNEL assays, in the SNpc of 6-OHDA injected rats (Zuch et al., 2000) Similar to dopamine-induced cell death, the level of p53 and Bax are also increased in models using 6-OHDA (Blum et al., 1997) Therefore, the mechanisms of cell death induced by both dopamine and 6-OHDA appear to be overlap This is not surprising since both compounds are very related in structure Like many models, 6-OHDA does not completely mimic all the clinical and pathological features of PD However, its natural existence and the potentially harmful effects of its presence still make it a good neurotoxin to use for the study of nigral degeneration 1.3.3 The Use of Rotenone in the Study of PD Rotenone has been one of the most successful neurotoxins which is able to induce almost all of the characteristics of PD Rotenone is a naturally occurring highaffinity complex I inhibitor It is an organic pesticide and is commonly used to kill nuisance fishes in lakes and reservoirs Owning to its extreme lipophilic nature, it can cross biological membranes easily and its movement is rapid and independent of transporters (as compared to MPTP/MPP+) As such, rotenone is a systemic complex I inhibitor which can act on all parts of the brain (Talpade et al., 2000) However, its administration causes only the selective degeneration of the striatal neurons (Betarbet et al., 2000) This is remarkable as it indicates that nigrostriatal neurons are particularly vulnerable to complex I inhibitors Therefore, exposure to such compounds could potentially be a factor in PD onset As for other neurotoxins, the exact mechanism in which it causes cell death is still unknown However, since it is a specific complex I inhibitor, evidences had surfaced which demonstrate its effects on inducing oxidative stress Chronic exposure of SH-SY5Y, a human neuroblastoma cell line, to rotenone over the course of four weeks greatly reduced the level of GSH In addition, there were observable increase in DNA oxidation and protein damage (Sherer et al., 2002) These rotenone-treated cells also showed apoptotic characteristic like the release of cytochrome-c and the activation of caspase-3 The same observations were demonstrated by another study using the same cell line (Kitamura et al., 2002) In this study, caspase-9, caspase-3 as well as caspase-12 were activated in the presence of rotenone Apart from caspase activation, other apoptotic characteristics e.g DNA fragmentation was also evident Thus, the mode of action of rotenone seems to suggest that a complex I-induced oxidative damage will subsequently lead to caspase activation and apoptosis Rotenone has been an excellent model for the study of PD based on three main observations Firstly, rotenone is a systemic complex I inhibitor This is in agreement with the observed loss of complex I activity in other types of cells i.e in the platelets of PD patients (Haas et al., 1995) Secondly, the specific and chronic degeneration of the SNpc induced by rotenone indeed recapitulate the nature of the progressive dopaminergic cell loss in PD patients Last but not least, rotenone-treated animals showed the presence of Lewy bodies, a cytoplasmic inclusion consisting of mainly the alpha-synuclein and ubiquitin proteins (Betarbet et al., 2000) This is one of the features that the other neurotoxins are unable to reproduce (Betarbet et al., 2002) Although rotenone has been able to reproduce most of the features of PD, its requirement for a chronic dosage regime into animal models is very capital- and labourintensive In addition, the major disadvantage of using rotenone is that not all animals and cells respond to its toxicity and develop SNpc leisions In summary, this neurotoxin can indeed recapitulate most of the features of PD Its niche in inducing Lewy bodies remained unparalleled However, the specific study of cell death would still require a neurotoxin which can cause a uniformed response This thus leaves a stage for the use of dopamine, 6OHDA, as well as MPTP/MPP+, in models for PD research 1.4 The Use of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyride (MPTP) in the Understanding of PD The use of MPTP in PD research has been extensive It was first discovered in 1979, when a group of young Californians addicted to a synthetic heroin analog was found to develop PD symptoms Post-mortem studies on the brains of these victims revealed the loss of cells in the substantia nigra similar to that found in PD (Davis et al., 1979) This led to the discovery of the neurotoxin MPTP MPTP was found to induce many of the biochemical and neuropathological changes that are observed in postmortem brains of PD patients Similarly, these changes were also observed in the striatal neuronal circuit of MPTP-treated animal models such as in monkeys and mice The changes include typical PD characteristics such as marked reduction of dopaminergic neurons and dopamine content The treated animals also manifest PD symptoms such as bradykinesis and muscle rigidity (Kopin and Markey, 1988; Langston, 1996) From then on, MPTP has been widely used to induce Parkinsonism in a variety of animal and cell models in order to study the death mechanisms behind the nigralstriatal cell death So far, the studies have 10 In conclusion, the death of MN9D cells challenged with MPP+ via the apoptotic pathway supports the growing evidences showing the same in dopaminergic cells of human and animal models The inhibition of the mitochondrial-mediated apoptotic pathway in this study was beyond expectation Thus, the generally accepted view that the mitochondria might be involved in the degenerative mechanism elicited by MPP+ or MPP+-like substances in the dopaminergic cells of PD 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