Neurotransmission, Oxidative Stress, and Coexistence of Neurotransmitters in Parkinson's Disease

Chapter 18

Neurotransmission, Oxidative Stress, and Coexistence of Neurotransmitters in Parkinson’s Disease G. Ali Qureshi

INTRODUCTION Parkinson’s disease (PD) is a neurodegenerative disorder of the central nervous system (CNS) that occurs most commonly in the elderly people evaluated by neurologists in their clinical practice. PD affects mainly, but not exclusively, the elderly – about 1% of the population affected is over 65 (1). In recent years, several genes that cause certain forms of inherited PD have been identified, and great progress has been made in elucidating their molecular mechanism. PD is a progressive neurodegenerative disorder with the cardinal features of bradykinesia, muscle rigidity, tremor at rest, and postural instability (2). PD occurs when nerve cells, or neurons, in an area of the brain known as the substantia nigra die or become impaired. It is now appreciated that PD is a basal ganglia disorder, and the symptoms result from a single transmitter deficit due to the loss of dopamine in the substantia nigra, pars compacta. The etiology of the disease is unknown (idiopathic), but epidemiological studies have suggested that exposure to toxins and viruses may predispose to the disease along with life events and the aging process. It is likely that about 70–80% of the nigrostriatal neurons have to cease functioning or die before the symptoms appear (3). Many brain cells of people with PD contain Lewy bodies – unusual deposits or clumps of the protein alpha-synuclein, along with other proteins. Researchers do not yet know why Lewy bodies form or what role they play in development of the disease. The small puzzle pieces provided by the multitude of researchers now seem to be crystallizing into a recognizable and useful model unified by a broad base of seemingly disparate etiologic factors including infectious agents, genetic predisposition, environmental factors, endotoxic factors, metabolic abnormalities, traumatic events, electromagnetic radiation exposure, antioxidants, sex hormones, pharmaceutical drugs, and others. However, other nerve cell populations using monoamine transmitters, such as the locus coeruleus containing noradrenaline neurons, are also affected (4–6). Pathologically, PD is associated with preferential degeneration of nigrostriatal dopamine neurons, but the degeneration also affects other central and peripheral nervous systems such as serotoninergic, noradrenergic, cholinergic and peptidergic pathways (7–9).

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© 2007 Elsevier B.V. All rights reserved.

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The etiology of neuronal death in neurodegenerative disease remains mysterious; however, great advances in both molecular genetics and neurochemistry have improved our knowledge of fundamental processes involved in cell death, including both excitotoxicity and oxidative damage (8). Although the importance of genetics in PD is increasingly recognized, most researchers believe environmental exposures increase a person’s risk of developing the disease. Even in familial cases, exposure to toxins or other environmental factors may influence when symptoms of the disease appear or how the disease progresses. There are a number of toxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, or MPTP (found in some kinds of synthetic heroin), that can cause parkinsonian symptoms in humans. Other, still-unidentified environmental factors also may cause PD in genetically susceptible individuals. Several lines of research suggest that mitochondria may play a role in the development of PD. Mitochondria are the energy-producing components of the cell and are major sources of free radical molecules that damage membranes, proteins, DNA, and other parts of the cell (9). The importance of decreased efficiency of mitochondrial oxidative phosphorylation activity is multifactorial. Perhaps the most important consequence of inefficient energy production is a change in the neuronal transmembrane potential. Under normal conditions, with adequate mitochondrial energy production, a normal transmembrane potential exists. The transmembrane potential has a profound effect on the activity of a specific receptor for the excitatory neurotransmitter glutamate. This receptor (NMDA receptor) under normal conditions of transmembrane electrochemical gradient (normal mitochondrial ATP production) is functionally blocked by a magnesium ion. When mitochondrial oxidative phosphorylation activity becomes depressed, alterations in the transmembrane potential relieve the magnesium block of the NMDA receptor, which, when stimulated by the excitatory neurotransmitter glutamate, causes influx of calcium into the cytosol (10–12). Several factors, such as inhibition of the mitochondrial respiration, generation of hydroxyl and nitric oxide radicals, and reduced free radical defense mechanisms causing oxidative stress, have been postulated to contribute to the degeneration of dopaminergic neurons (13–15). In the past decade or so, a convincing link between oxidative stress and degenerative conditions has been made and, with the knowledge that oxidative changes may actually trigger deterioration in cell function, a great deal of energy has been focused on identifying agents that may have possible therapeutic value in combating oxidative changes with antioxidants, which may prevent or reduce the rate of progression of this disease (16). On the other hand, the discovery of several genetic mutations associated with PD raises the possibility that these or other biomarkers may help identify persons at risk of PD (15). Other research suggests that the cell’s protein disposal system may fail in people with PD, causing proteins to build up to harmful levels and trigger cell death. However, the precise role of the protein deposits remains unknown. Some researchers even speculate that the protein buildup is part of an unsuccessful attempt to protect the cell. While mitochondrial dysfunction, oxidative stress, inflammation, and many other cellular processes may contribute to PD, the actual cause of dopamine cell death is still undetermined (17). The treatment of choice is still mainly symptomatic, either by means of substances acting on the dopaminergic system, such as l-DOPA and dopamine (DA) agonists, or by means of drugs that modify the metabolism of l-DOPA or DA with MAO B and

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COMT inhibitors (11). Because of its diagnosis at the later stage, the actual treatment strategies have pitfalls and caveats. The discovery of l-DOPA marks a major milestone in modern neuropharmacology. Among so many pharmacological therapies adopted in the past two or three decades, the most effective therapy is with l-DOPA, which is most successful during the first years of PD. However, l-DOPA shows that more than 80% of PD patients after 5–10 years show On and Off phenomena in the PD patients with dyskinesia. Its limitation is most noteworthy in their advanced stages, where it is unable to improve motor and nonmotor parkinsonian features, giving visible side effects, and it does not halt PD progression. Levodopa reduces morbidity and mortality in PD. The FDA approved carbidopa–levodopa in 1982 as PD monotherapy. Levodopa, the precursor of dopamine, is converted by dopa decarboxylase in the peripheral nervous system and CNS. Levodopa can cross into the brain from the blood–brain barrier (BBB), where the action is most needed for parkinsonism. However, once levodopa is converted to dopamine in the periphery, dopamine cannot penetrate the BBB. Most levodopa is metabolized in the periphery (only 1% penetrates the brain) (18). The dopamine remaining in the periphery can cause systemic side effects such as nausea and vomiting (80%), anorexia (50%), postural hypotension (30%), and arrhythmias (10%). Carbidopa is added to maximize levodopa in the brain and to minimize systemic side effects. Despite disadvantages, l-DOPA remains the gold standard for the treatment of PD due to its impressive efficacy on the motor symptoms and its lower cost as compared to other treatments. Several motor features typically do not respond to l-DOPA. On the contrary, it tends to deteriorate speech, gait, posture, and balance over time. In addition, l-DOPA therapy tends to aggravate nonmotor functions, such as hallucinations, cognitive impairment, and orthostatic hypotension. Recently, a study (19) reported that motor fluctuations and dyskinesias could be minimized by using the lowest possible l-DOPA dosage throughout the several years of treatment. This is one of the main reasons why the development of dopamine agonists was demanded as a new treatment strategy (20,21). Various drugs such as Bromocriptine, Lisurid, and Pergolid are introduced; however, these drugs have limited efficiency in the long-term treatment of de novo patients. Furthermore, the patients had more side effects, and the clinical outcome was poorer than when patients were treated with l-DOPA (20,21). Other studies of early combination of l-DOPA with dopamine agonists show reduced dyskinesia and fluctuations in the long term (22,23). Mostly the treatment is age related which contains either dopamine agonist or combined l-DOPA with dopamine agonists are employed. However, one recent study (24) could not find any relationship between the age and the treatment type with regards to the clinical outcome. This chapter deals with CSF analysis of small peptides such as substance P (SP), neuropeptide Y, and both tetrapeptides and octapeptides of cholecystokinin. The correlation between catecholamines and small peptides is also attempted. These levels are reported with the assumption that CSF concentration reflects brain or spinal cord concentrations and perhaps synaptic activity, since CSF is in constant exchange with the extracellular fluid of the CNS. Based on the fact that most of the transmitters in the brain are present in the CSF, and because alterations in the concentration of transmitters in the CSF can alter the same transmitters in the brain of man (25), the results are compiled in PD patients (On and Off groups) and compared with healthy subjects.

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MATERIAL AND METHODS Patients, healthy subjects, and routine analysis A total of 36 patients (15 women and 21 men) with PD were recruited from the Department of Neurology, Huddinge University Hospital, Stockholm. After approval from the Ethics Committee at the Karolinska Instituet, Stockholm, all the patients gave written informed consent to participate. Eighteen patients, each at different stages of illness (Hoehn and Yar range 2–4), were on l-DOPA dose (250 mg/day) and had taken it in combination with other drugs, with positive response in their motor activities (PD On group). Eighteen PD patients who had been treated for 6–8 years on a similar drug therapy and had shown severely negative response (PD Off group) were included, and they showed motor fluctuation. Both groups were monitored one week before the samples of blood and CSF were taken, all food and fluid intakes were similar in both groups, and exact dose time was adhered to. Blood and CSF were taken early in the morning between 6–8 AM in a fasting condition. Eighteen healthy individual volunteers from among the Karolinska Instituet employees were included, and their blood and CSF were collected under conditions similar to those of the patient groups. None of the healthy controls were on medication for six months. About 10–12 ml cerebrospinal fluid (CSF) was collected from each PD patient and healthy control in the sitting position at the L4–L5 levels. Blood samples were collected by venipuncture. Serum albumin, CSF albumin, and IgG were determined using Hitachi 737 Automatic Analyzer (Naka Works, Hitachi Ltd., Tokyo, Japan). CSF and serum samples were kept at −80◦ C if not analyzed immediately. Clinical data on both PD groups along with healthy controls are shown in Table 1. Analysis of amino acids, catecholamines, tryptophan Amino acid analysis was performed based on the precolumn derivatization of OPA with amino acids, as previously described (26). The reproducibility for each amino acid was >98% with this method. Data are presented as mean ± SEM. Differences in concentration of vitamin B12 and homocysteine were analyzed with ANOVA, and group comparison was done with t-test. A p-value of less than 0.05 was considered significant. The concentrations of DA, homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), NA, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT),

Table 1. Clinical data on healthy controls and PD patients in both On and Off groups Patients

n

Females

Age (years)

CSF-Albumin

CSF-IgG

IgG-Index

Controls PD (On) PD (Off)

18 18 18

7 8 9

53 ± 7 72 ± 12 74 ± 14

218 ± 17 332 ± 42* 313 ± 37*

34 ± 4 51 ± 7** 44 ± 6*

0.44 ± 0.01 0.43 ± 0.01 0.42 ± 0.01

*p < 0.05, **p < 0.01

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and 5-hydroxyindoleacetic acid (5-HIAA) were measured as previously described (27). Measurements were also made in serum; however, since no differences were found, these will not be considered further. On and Off phenomena in PD Genetic, toxic, nutritional, traumatic, and lifestyle models have been proposed as playing major roles in the etiology of various neurodegenerative diseases. A model in which genetically predisposed individuals manifesting demonstrable hepatic detoxification flaws enhancing the neurotoxic effects of xenobiotics leading to neuronal mitochondrial failure unifies these seemingly disparate theories into an integrated model of neurodegenerative diseases. PD is a neurodegenerative disease that at present has no cure, and, despite the variety of pharmacological and surgical treatment options (28–30), it usually results in severe disability. In Europe, age-adjusted prevalence rates of PD have been estimated at 1.6 per 100 of the population, with a steady increase in older groups, up to 3.5–3.6 in people aged 80 years and older (31). Similar estimates have been reported for the US (32). In view of the increasing number of elderly people in developed countries, the prevalence of PD is expected to increase as well. Characteristics of PD include motor dysfunction, autonomic disturbances, and mental alteration. Motor disturbance is characterized by small handwriting (micrographia), pill-rolling finger tremor, decreased facial expressions (hypomimia), soft speech (hypophonia), decreased walking movement (shuffling gait), and decreased blink rate (33–35). As PD advances, patients develop dementia, confusion, psychosis, and sleep disturbance. Cognitive function declines with progression of the disease. PD patients with major depression may show a greater decline than patients with minor or no signs of depression (36). Diagnosis is based on symptoms. Mild, early disease may be difficult for doctors to diagnose because it usually begins subtly. Diagnosis is especially difficult in older people, because aging can cause some of the same problems as PD, such as loss of balance, slow movements, muscle stiffness, and stooped posture. No tests or imaging procedures can directly confirm the diagnosis. However, computed tomography (CT) and magnetic resonance imaging (MRI) may be performed to look for a structural disorder that may be the cause of the symptoms. Hoehn and Yahr developed a system to classify the disease into five different stages (37). This allows one to determine disability and the rate at which the disease is progressing. Patients in stages I or II have mild symptoms, requiring minimal treatment. Patients in stages III, IV, or V have many disabilities, such as bilateral postural instability and bed restriction, and require patient-specific treatment (38). The diagnosis of PD is likely if drug treatment for the disease results in improvement. Treatment with levodopa can produce dramatic improvement in people with PD, but people with parkinsonism due to another disorder usually do not improve. PD is associated with depression, demoralization, anxiety, and psychosis. Depression in PD is overlooked because of the overlap between motor and mental slowing. Treatment includes psychotherapy, pharmacotherapy, and electroconvulsive therapy. Several of the newer antidepressants are effective in patients with PD, as is electroconvulsive therapy. Anxiety is common in patients with PD and can interfere with their response to treatment. Psychosis can occur

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with any of the drugs used to treat PD. Some of the atypical neuroleptics, as well as electroconvulsive therapy, can be helpful. Because PD is a chronic condition, the disease burden increases as the illness progresses, due to the appearance of both disease- and drug-related problems, resulting in the extensive utilization of both health and community services. When the brain initiates an impulse to move a muscle (for example, to lift an arm), the impulse passes through the basal ganglia (collections of nerve cells located at the base of the cerebrum, deep within the brain). The basal ganglia help smooth out muscle movements and coordinate changes in posture. Like all nerve cells, those in the basal ganglia release chemical messengers (neurotransmitters) that trigger the next nerve cell in the pathway to send an impulse. The main neurotransmitter in the basal ganglia is dopamine. Its overall effect is to increase nerve signals to muscles. In PD, nerve cells in part of the basal ganglia (called the substantia nigra) degenerate, reducing the production and number of connections between nerve cells in the basal ganglia. As a result, the basal ganglia cannot smooth out movements as they normally do, leading to tremor, incoordination, and slowed, reduced movement (bradykinesia). PD is an inexorably progressive disorder that worsens over time. The rate of nigral cell death is not exactly known, but neuroimaging techniques estimate that cell death occurs at a rate of approximately 10% per year (39). PD symptoms are considered to be the consequence of an imbalance between stimulatory and inhibitory impulses in the extrapyramidal system, and this is mainly due to DA transmission in the nigrostriatal pathway. Disease progression mainly affects presynaptic terminals by reducing not only their buffer capacity but also their feedback control on striatal neurons (40). Various studies suggest that PD can be dependent on exposure to pesticide, herbicide, well water, and rural living, and not on cigarette smoking and caffeine consumption (39–41). The diagnosis of PD generally relies on the clinical observation of the combination of four cardinal motor signs, namely, tremor, rigidity, bradykinesia, and balance impairment or postural instability (9). These symptoms, especially the first three, are typically improved by dopamine replacement therapies, and a positive response to l-DOPA is mandatory for the diagnosis of PD. However, not all motor features in PD are adequately controlled with dopaminergic medication. Furthermore, PD is not just a motor disorder, and dysfunctions of autonomic, cognitive, and psychiatric systems frequently accompany the classic motor features of PD (43). These nonmotor features frequently represent an important source of disability for PD patients and severely impact their quality of life. There is no specific cure for PD, and there are limitations in current PD therapy (42). However, there are several treatments available to alleviate symptoms of the disease, such as pharmacological therapy (43) and surgical interference (44). Levodopa is the gold standard for treating the symptoms of PD. However, patients taking levodopa may develop motor complications that limit the use of levodopa (45). Some question whether levodopa treatment should be postponed until patients fail to respond to other therapies. Some clinicians propose initial use of dopamine agonists to prevent motor complications caused by the long-term use of levodopa (46,47). Health care providers should decide which treatment is most appropriate for each patient. The treatment of choice is still mainly symptomatic, either by means of substances acting on the dopaminergic system, such as l-DOPA and dopamine (DA) agonists, or by means of drugs that modify the metabolism of l-DOPA or DA with MAO B and COMT inhibitors (11). The most successful drug therapy is still l-DOPA,

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which is a precursor in dopamine biosynthesis. Mortality has been nearly normalized, and l-DOPA is still considered the most effective agent for the treatment of PD. However, disabling motor complications occur in the late stages of PD, with the most common effect that includes onset of On and Off phenomena and abnormal involuntary movements called dyskinesia (11). Long-term use of carbidopa–levodopa can cause some potentially disabling complications, including dyskinesia (involuntary muscle movements or jerky motions), blepharospasm (involuntary contraction of eyelid muscle), motor fluctuations, and hallucinations (48) Motor fluctuations – random conversion from a mobile state to an immobile state and vice versa – are also known as the “On–Off” phenomenon (49). They are associated with fluctuating responses to levodopa. Patients respond to the drug during the “On” state and are unresponsive during the “Off” state. Most PD patients treated with l-DOPA develop fluctuation in motor performance. After 3–5 years of treatment, one-third; after 5–7 years, about half; and after 10–12 years, nearly all patients suffer from the motor fluctuation (22). Furthermore, nonmotor symptoms such as pain, fatigue, anxiety, and depression are often seen in PD patients treated with l-DOPA for a long time (>10 years) than are seen in patients with classical motor disturbances (50,51). And, therefore, it has been suggested that l-DOPA therapy be delayed as long as possible (52). Most cases of PD, however, appear to be sporadic, and these are likely to represent an interplay between both genetic and environmental factors. To date, the main risk factors for developing PD in an individual, apart from biochemical brain defects, are increasing age and presence of another affected family member (53). It is also proposed that the earlier the age of PD onset, the greater the likelihood that genetic factors play a dominant role. Asians and African blacks have the lowest reported incidence of the disease. To what extent this reflects environmental or genetic differences or differences in ascertainment is not clear, but the prevalence of Lewy bodies in the brains of Nigerians is similar to that of Western populations (54). Several multigenerational families have been described with pathologically confirmed PD, although in these there are usually atypical features such as rapid rate of progression or a high frequency of dementia (55). Mutations in 1 exon of the alpha-synuclein gene were recently discovered in a large Italian and three smaller Greek families that may have been related (56), and in another exon of the same gene in a German family (57). However, the disease is rapidly progressive and has a young age of onset in these families. Moreover, mutations in this gene have not been found in other families and sporadic PD. In young-onset PD, the frequency of mutations in the PARKIN gene is high when a first-degree family member is also affected (58). Mutations in this gene were originally described in autosomal recessive Japanese pedigrees with prominent sleep benefit, sensitivity to extrapyramidal side effects of l-DOPA, and absence of nigral Lewy bodies in pathologically studied cases. A susceptibility locus on chromosome 2 in familial PD with features more closely resembling sporadic PD has been described in six families with autosomal dominant inheritance with low penetrance (59). Pharmacokinetics of L-DOPA and the BBB l-DOPA is transported across the intestinal mucosa and the BBB by the large neutral amino acid (LNAA) transport system, which means that some amino acids competitively inhibit

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l-DOPA membrane transport (60). Doses of l-DOPA taken with a meal rich in proteins will be less efficacious than doses taken on an empty stomach (61). By restricting most of the daily protein intake to evening meals, daytime plasma l-DOPA levels are more predictable, and motor performance is improved. Hence, it is concluded that plasma l-DOPA levels predict motor response to a greater extent under controlled conditions regarding diet and medication (62). Under healthy conditions, the BBB regulates the entry of any drug or endogenous compound to peripheral organs. However, the barrier functions of BBB can change dramatically during various CNS diseases. The most common consequence under inflammation and bacterial disorders is an alteration of BBB permeability (63). In addition, antidiuretic hormone secretion and accumulation of toxic substances also affect BBB transport properties (64). On the other hand, l-DOPA is susceptible to a large number of pharmacokinetic interactions (65), and it has a short half-life. It produces remarkable blood fluctuations of the drug with unimportant consequences in early stages of the disease, but not so at the later more advanced stages. This is due to the complicated kinetics in the brain as compared to the kinetics in the blood. This makes it clear that PD is an evolving disease not only from a neuropathological and pharmacodynamic point of view, but also from a pharmacokinetic one (66). This may be the reason why there are different responses to l-DOPA at the early stage (On phenomena) and at the advanced stage (Off phenomena), where the number of dopaminergic neurons are destroyed with time (67). Although the mechanism responsible for motor fluctuations is unknown, studies have supported the storage hypothesis. Usually in the early stages of the disease, patients have sufficient presynaptic dopaminergic neurons to store and release excess dopamine supplied by dopamine therapy in the striatal region. The dopamine storage allows the brain to control the fluctuating level of neurotransmitter, permitting normal motor function. However, as the disease progresses, striatal dopamine neuronal terminals decrease, decreasing the brain’s capacity to store dopamine and shortening the medication’s duration of action over time. With declining storage ability, the body is vulnerable to changing levels of dopamine (especially from external sources, e.g. levodopa) in the brain, which cause motor instability. Dyskinesia occurs when the dopamine level is too high, while bradykinesia or freezing (immobility of the body) results when the level is very low (48). In order to study these changes, CSF obtained by the lumbar puncture technique is the most important and widely used diagnostic tool in evaluating the levels of neurotransmitters and their metabolites. Lumbar CSF neurochemical measurements aim to serve as indirect in vivo markers of human central neurotransmission, and these markers can be used in the diagnosis of neurodegeneration processes. Free radicals and oxidation stress in PD All aerobic organisms are susceptible to oxidative stress simply because semireduced oxygen species, superoxide and hydrogen peroxide, are produced by mitochondria during respiration. The exact amount of ROS produced is considered to be about 2% of the total oxygen consumed during respiration, but it may vary depending on several parameters. Brain is considered abnormally sensitive to oxidative damage, and, in fact, early studies demonstrating the ease of peroxidation of brain membranes supported this notion. It is

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interesting to note that in PD, mitochondrial dysfunction leading to excessive free radical production and oxidative tissue damage seems to be confined to the brain, despite the fact that the underlying mitochondrial abnormality is systemic. Indeed, mitochondrial defects in platelets in PD (50% deficiency in complex I activity) have been well described (66). This may be explained by the unique susceptibility of the brain to mitochondrial dysfunction and the resultant excessive free radical production, since the brain uses approximately 20% of the total O2 consumption (while representing only 2% of the body’s weight). Thus, being so highly metabolic, brain tissue generates more oxyradicals. Second, neurons are post-mitotic. This allows accumulation of oxidatively damaged DNA, proteins, and lipids compared to cells that retain the property to undergo mitosis. Third, compared to other highly metabolic tissues, the brain has relatively low levels of protectant antioxidant enzymes and small-molecule antioxidants (67,68). In normal circumstances, cellular defenses protect against damaging reactive oxygen species – hydrogen peroxide and free radicals such as superoxide, peroxyl, nitric oxide, and hydroxyl radicals. These compounds react with lipids, proteins, and DNA, altering structure and function. This oxidative stress is increased in PD. Increasing iron in pars compacta of the substantia nigra and depleted reduced glutathione (one of two major free radical scavengers in the brain, the other being glutathione peroxidase) are believed to be the factors contributing to increased oxidative stress in PD. Dopamine turnover can also produce oxidant stress as dopamine oxidation leads to the formation of hydrogen peroxide (69). This is one of the reasons why levodopa (which is converted to dopamine) has been regarded by many as potentially harmful to the remaining nigral dopaminergic cells (70), although it is debated whether this is important in practice (71). Most free radicals are unstable species due to one or more unpaired electrons that can extract an electron from neighboring molecules, leading to oxidative damage. The role of free radicals in cell death induced by activation of EAA receptors is an area of expanding interest (72). Brain is known as a highly oxygenated organ. Oxygen-centered free radicals are the main types of radicals formed in neurons as accidental by-products of metabolism or as selectively generated species commonly known as reactive oxygen species (ROS). There is increasing evidence that free radical nitric oxide (NO) plays an important role in pathophysiology of a variety of CNS disorders (73–75). ROS are very important mediators of cell injury and death. Not only are these highly reactive chemical species important in the imaging process, but they are also directly or indirectly involved in a wide variety of pathological conditions (76–80). The generation of ROS in excessive amount is enough to overwhelm normal defense mechanisms, and can result in serious cell and tissue damage. Nearly all the major classes of biological molecules can result in their structural and biological activity destruction, especially membrane lipids, which are most susceptible. Among the ROS, superoxide anion (O•2 ) and nitric oxide (NO• ) are the most studied free radicals, and the mammalian brain may be exceptionally vulnerable to oxidative stress through the attack of these radicals (81). Nitric oxide is shown implemented in various neurodegenerative disorders (82,83). Although nitric oxide itself is a free radical due to its unpaired electron, it is not thought to participate in any significantly damaging chemical reactions. However, when reacting with superoxide anion, the extremely reactant and potent oxidant peroxynitrite (ONOO) is formed. This reaction is approximately three times faster than the reaction dismutating superoxide to form

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hydrogen peroxide catalyzed by superoxide dismutase (SOD) (84). Peroxynitrite has been implicated in a variety of damaging intraneuronal events, including DNA strand breaks, DNA deamination, nitration of proteins including superoxide dismutase, and damage to mitochondrial complex I, complex II, and mitochondrial aconitase. In addition, nitric oxide itself also specifically damages mitochondrial complex I (85). It is also known that enhanced Glu release increases free radical, nitric oxide (NO) production, causes DNA damage, energy depletion in neurons, and neuronal death in PD (80). This hypotheses is also supported by our recent study (80), which showed high levels of arginine and NO in patients with PD. Table 3 shows CSF levels of arginine (Arg), the precursor of NO and nitrite (a metabolite of NO) in healthy controls and On and Off groups of PD patients, where both Arg and NO are significantly increased in both PD groups. Elevated levels of nitric oxide synthase have been found in the brains of patients with multiple sclerosis (85). The role of nitric oxide in mediating neuronal damage in cerebral ischemia is also the subject of intense research. Again, the operative model recognizes excessive glutamate stimulation of the NMDA receptor in cerebral ischemia with elevation of intracellular calcium and induction of nitric oxide synthase, raising intraneuronal nitric oxide (81). Several investigators have repeatedly shown a modest (30–40%) decline in mitochondrial complex I activity in platelets, muscle, and discrete brain regions of patients with PD (86,87). Oxidative stress, by damaging mitochondria, may reduce complex I activity. The mitochondrial respiratory chain, particularly when impaired, is a potent source of free radicals, reduced complex I activity may also generate oxidative stress and deplete reduce glutathione. Two independent studies have suggested that the origin of complex I deficiency PD is the mitochondrial DNA (88), except in rare cases (89) where specific mitochondrial DNA mutations have not been found in parkinsonian conditions. In our recent studies (82), a linear correlation between NO and Glu for both these groups was found (Fig. 1).

5

Controls PD (On) PD (Off)

4

NO2

3 2 1 0 0

10

20 GLU

30

Fig. 1. Correlation between nitrite and glutamic acid in CSF of PD patients (On and Off status) and healthy subjects.

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3

2 HC

Controls PD (On) PD (Off) 1

0 0

1

2 NITRATE

3

4

Fig. 2. Correlation between homocysteine (HC) and nitrite in CSF of PD patients (On and Off status) and healthy subjects.

Neuroleptics are the major class of drugs used to treat PD; however, these drugs are associated with wide variety of extrapyramidal side effects, including tardive dyskinesia (90). Numerous reports have indicated that induction of free radicals in neuroleptic drug treatment increase the free radical production and causes structural damage that eventually lead to oxidative stress (91,92). Besides, NO exposure causes cobalamin deficiency resulting from its inactivity, which is known to produce impaired neurological functions (93–95). In our patients, indeed, deficiency of vitamin B12 with elevation in the concentration of homocysteine, glutamic acid, and the correlation with the free radical nitric acid was shown. Figure 2 shows the correlation between homocysteine and nitrate content in CSF, suggesting that neurotoxicity caused by homocysteine and glutamic acid in the brain is directly related to oxidative stress caused by the accumulation of free radicals such as nitric oxide. Naturally occurring molecules such as bioflavonoids, polyphenols, vitamins, terpenes, alkaloids, and enzymes constitute the barrier that plants, animals, and humans used to oppose the damaging free radicals and reveals the effective natural remedy in facing several radical-related diseases. The mechanism of the damage that free radicals produce on biomolecules such as on polyunsaturated fatty acids and nucleic acids is known. Thus, it is essential to maintain the balance between prooxidants and antioxidants in vitro and in vivo. In case of oxidative stress damaging the free radicals, both reactive oxygen species and reactive nitrogen species are removed or converted into harmless species. Biological systems in humans have developed a comprehensive array of defense mechanisms to protect against free radicals. These include enzymes to decompose peroxides, proteins to sequester transition metal ions, and a range of compounds such as antioxidants to scavenge free radicals. Oxidative stress is caused by the accumulation of free radicals and is defined as a disturbance in prooxidant and antioxidant balance in favor of the former leading to potentially damaging reactions with biological molecules (82).

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The results indicate a possible participation of oxidative stress in the neuropathology of PD patients, especially during the crisis, i.e. in Off situation, when the metabolites are highly increased, and to a point where the use of antioxidant drugs as a possible adjuvant therapy to improve the neurological status of PD patients and to prevent sequelae. An unbalanced overproduction of ROS may give rise to oxidative stress, which can induce neuronal damage, ultimately leading to neuronal death by apoptosis or necrosis. Oxidative stress is a ubiquitously observed hallmark of neurodegenerative disorders. Neuronal cell dysfunction and cell death due to oxidative stress may causally contribute to the pathogenesis of progressive neurodegenerative disorders, such as Alzheimer’s disease (AD) and PD, as well as acute syndromes of neurodegeneration, such as ischemic and hemorrhagic stroke (96–100). A large body of evidence indicates that oxidative stress is involved in the pathogenesis of AD and PD. An increasing number of studies show that nutritional antioxidants (especially vitamin E and polyphenols) can block neuronal death in vitro, and may have therapeutic properties in animal models of neurodegenerative diseases including AD, PD, and ALS. Moreover, clinical data suggest that nutritional antioxidants might exert some protective effect against AD, PD, and ALS. Hantraye and associates in Orsay, France, published evidence in 1996 demonstrating that pretreatment of baboons with the nitric oxide synthase inhibitor 7-nitroindazole (101) completely prevented the induction of parkinsonism in baboons exposed to MPTP. These researchers demonstrated that inhibiting nitric oxide synthase “protected against profound striatal dopamine depletion and loss of tyrosine-hydroxylase-positive neurons in the substantia nigra” and “protected against MPTP-induced motor and frontal-type cognitive deficits” (101). Furthermore, neuroprotective antioxidants are considered a promising approach to slowing the progression and limiting the extent of neuronal cell loss in neurological disorders. The clinical evidence demonstrating that antioxidant compounds can act as protective drugs in neurodegenerative disease, however, it is still relatively scarce. The major challenges for drug development are the slow kinetics of disease progression, the unsolved mechanistic questions concerning the final causalities of cell death, the necessity to attain an effective permeation of the BBB, and the need to reduce the high concentrations currently required to evoke protective effects in cellular and animal model systems. Finally, an outlook as to which direction antioxidant drug development and clinical practice may be leading to in the near future will be provided. Antioxidants of widely varying chemical structures have been investigated as potential therapeutic agents. However, the therapeutic use of most of these compounds is limited, since they do not cross the BBB. Although a few of them have shown limited efficiency in animal models or in small clinical studies, none of the currently available antioxidants have proven efficacious in a large-scale controlled study. One of the most promising agents for up-regulation of mitochondrial function is Coenzyme Q-10. Coenzyme Q-10, in addition to having free radical scavenging properties, is known to play a pivotal role in transporting electrons in the mitochondria for ATP production. The usefulness of Coenzyme Q-10 in specific mitochondrial myopathies has been well described. Bresolin and coworkers in Milano, Italy, have described enhanced mitochondrial activity as evidenced by reduction of serum lactate and pyruvate following standard muscle exercise with generally improved neurological functions in Kearns Sayre syndrome and chronic progressive external ophthalmoplegia (102). Therefore, any novel antioxidant molecules designed

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as potential neuroprotective treatment in acute or chronic neurological disorders should have the mandatory prerequisite that they can cross the BBB after systemic administration. In addition, Ginkgo biloba is known to be involved in such diverse processes as homeostasis of inflammation, reduction of oxidative stress, membrane protection, and neurotransmission modulation (103). Recently, alpha lipoic acid has emerged as one of the most promising agents for neuroprotection in neurodegenerative diseases. This potent antioxidant demonstrates excellent BBB penetration. It acts as a metal chelator for ferrous iron, copper, and cadmium, and also participates in the regeneration of endogenous antioxidants including vitamins E, C, and glutathione. Although no large clinical evaluation of the usefulness of alpha lipoic acid in neurodegenerative diseases has as yet been published, an excellent review in a paper entitled “Neuroprotection by the metabolic antioxidant alpha lipoic acid” by Packer and co-workers in Frankfurt, Germany, provides enough justification for strong consideration of alpha lipoic acid as a neuroprotectant for neurodegenerative conditions (104). Monoamines and their metabolites Catabolism of tryptophan and tyrosine in relation to the isoprenoid pathway was studied in neurological and psychiatric disorders. The concentrations of tryptophan, quinolinic acid, kynurenic acid, serotonin, and 5-hydroxyindoleacetic acid were found to be higher in the plasma of patients with all these disorders, whereas those of tyrosine, dopamine, epinephrine, and norepinephrine were lower. It is known that the level of free tryptophan in the blood can influence the transport of tyrosine across the BBB into the brain and vice versa, since both these amino acids share the same transport systems and compete with each other. The estimation of monoamines and indoleamines have been shown to be helpful for the diagnosis and interpretation of various neurological and psychiatric disorders (105–107). Both noradrenaline (NA) and dopamine (DA) are synthesized from the amino acid tyrosine, derived from food intake or protein breakdown. The main metabolites of DA are homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), and of NA, 3-methoxy-4-hydroxyphenylglycol (MHPG). In PD patients, the CSF NA and its metabolite, MHPG, are increased in both On and Off groups, whereas DA and its metabolites, DOPAC and HVA, are significantly decreased in both groups. Figures 3 and 4 show CSF levels of DA, NA, and their metabolites in On and Off PD patients and are compared with healthy controls. A single dose of l-DOPA gives a consistent response to motor activity in On situation, whereas when the disease progresses within a few years, the response duration gets shorter, which is when DA and its metabolites are further decreased in the Off situation due to the therapeutic window being narrowed. Apart from catecholamines, serotonin (5-HT), which is synthesized from the amino acid tryptophan, plays very important role in CNS. The aromatic amino acids L-TRYPTOPHAN and L-TYROSINE are the most important in this respect. L-TRYPTOPHAN is the precursor of not only serotonin, a well-known neurotransmitter, but also of two other neuroactive substances, quinolinic acid and kynurenic acid. L-TYROSINE is the precursor of dopamine and other catecholamines. Alteration in tryptophan catabolism has been reported in neurodegenerative disorders such as

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300

DOPAC HVA DA

*

pmol/l

** 200

100 *

**

0 CONTROLS

PD (ON)

PD (OFF)

Fig. 3. CSF levels of dopamine (DA) and its metabolites, HVA, and DOPAC in PD patients (On and Off status) and healthy subjects.

HMPG NA 200 * * pmol /l

*

*

100

0

Controls

PD (ON)

PD (OFF)

Fig. 4. CSF levels of norepinephrine (NA) and its metabolite, HMPG, in PD patients (On and Off status) and healthy subjects.

Huntington’s disease (108). Very few reports are available on tyrosine metabolism in these disorders. Morphine, an alkaloidal neurotransmitter, is synthesized from tyrosine (109). CSF levels of tryptophan and its metabolites 5-HIAA and 5-HT are shown in Table 2 in PD patients. Recently, the presence of endogenous strychnine and nicotine has been reported in the brains of rats loaded with tryptophan (110). 5-HT is metabolite to 5-hydroxy indole acetic acid (5-HIAA). The estimation of 5-HT is related to depression, and Table 2 shows the levels of tryptophan, 5-HT, and 5-HIAA, which are significantly decreased in both groups as they are known to be depressed and inactive (110).

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Table 2. CSF levels of tryptophan and its metabolites in healthy controls and patients with PD On and Off groups. All values are expressed as mean ± SEM in pmol/l Patients

TRP

5-HT

5-HIAA

Healthy controls PD (On) PD (Off)

2115 ± 105 1635 ± 99** 1378 ± 78***

9.7 ± 1.2 7.3 ± 0.85* 4.8 ± 0.52***

296 ± 51 241 ± 39* 202 ± 23**

*p < 0.05, **p < 0.01, ***p < 0.001

Cobalamin and its role in PD It is also known that the free radical nitric oxide NO interacts with vitamin B12 , resulting in selective inhibition of methionine synthase a key enzyme in metabolism of methionine and folate. Thus, NO may alter one-carbon and methyl group transfer that is most important for DNA purine and thymidylate biosynthesis. Our results indeed show the correlation between nitrite and homocysteine, suggesting that both these parameters are interrelated (83). Although the mechanisms responsible for the neurological lesion by vitamin B12 deficiency are less well understood, vitamin B12 analogues including methylcobalamin (MCbl) have been widely used in therapy of neurological diseases (111). Vitamin B12 was shown to improve memory, emotional function, and communication ability in Alzheimer’s patients. MCbl is an active coenzyme of vitamin B12 analogues that is essential for cell growth and replication (112). In fact, detection of vitamin deficiency in humans is a problem to solve in clinical area, because data on its levels in blood do not always correlate with CSF levels. However, there is now good evidence that the majority (>90%) of the patients who are vitamin B12 deficient accumulate high levels of methylmalonic acid and homocysteine in blood. Hence, both these substances can be considered better markers for clinical diagnosis of vitamin B12 deficiency (112). Table 3 shows the CSF levels of cobalamin and homocysteine in PD patients (On and Off groups), which are statistically compared with healthy controls. Cobalamin is

Table 3. CSF levels of vitamin B12 and homocysteine in healthy controls and patients with PD On and Off groups. All values are expressed as mean ± SEM in µmol/l Patients

Vitamin B12

Homocysteine

Healthy controls PD (On) PD (Off)

0.079 ± 0.006 0.059 ± 0.005** 0.041 ± 0.003***

1.09 ± 0.096 1.56 ± 0.130** 1.89 ± 0.21***

**p < 0.01, ***p < 0.001

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significantly decreased, and an increase in homocysteine is observed in both On and Off groups of PD. NO toxicity due to its interaction with a vitamin-B12 -dependent enzyme, i.e. methionine synthase, produces hematological and, less frequently, neurological symptoms (113). NO radical is also known to oxidize active cob(II)alamin to inactive cob(III)alamin. As Cbl in the form of reduced MCbl is required as cofactor for methionine synthase, exposure to NO radical causes rapid inactivation of this enzyme (114). Hence, the inactive Cbl is excreted, so that repeated exposure to NO radical results in depletion of body Cbl stores, with reduced AdoCbl. The activity of the AdoCbl-dependent enzyme methylmalonic acid CoA (MMCoA) is also affected, resulting in impaired neurological functions. NO radical interaction with cobalamin results in the liberation of hydroxyl free radicals responsible for inactivation of methionine synthase (115). In patients with cardiac disorders, the high levels of blood homocysteine translate into a significant increase in hardening of arteries, known as arteriosclerosis. For a high-risk person suffering from moderate or severe arteriosclerosis, this increase in homocysteine could be enough to trigger a heart attack (116). It appears that these patients are advised to be given supplementary vitamins B6 , B12 , and folic acid, along with antioxidants such as vitamins C and E where the level of homocysteine can be reduced, and, in addition, it can also protect the blood from clotting (116). Pharmacological strategy in PD There should be two major strategies for trying to improve PD patients on l-DOPA therapy. One involves developing specific therapies for each of the problems that is unresponsive or aggravated by l-DOPA. These may include symptomatic treatments for motor fluctuations or dyskinesias, antidementia or antipsychotic agents, and drugs to control orthostatic hypotension, impotence, constipation, and abnormal daytime somnolence. A second strategy should be to devise diagnostic methods in identifying PD in early stages, blocking disease progression with efficacious and safe neuroprotective agents to prevent the disease from reaching an advanced stage in which new features develop that do not respond to current treatment, i.e. the Off phenomena. More recent therapeutic interventions, including DA agonists, MAO-B inhibitors, COMT inhibitors, and modern functional surgery such as deep-brain stimulation, have been developed to help control l-DOPA therapy shortcomings. Although helpful, such complementary interventions are not fully safe or efficacious (117). Symptomatic orthostatic hypotension is present in about 20% of PD patients and can be worsened by dopaminergic drugs (118). Constipation, neurogenic bladder with urinary frequency, urgency and incontinence, sexual dysfunction, and abnormal sweating and salivation are also frequent (119). Levodopa should not be taken with monoamine oxidase type A inhibitors (MAOI-A), such as isocarboxazid, phenelzine, and tranylcypromine, or agents with MAOI-like activity (isoniazid, linezolid, or procarbazine) due to an increased risk of hypertensive crisis (120). Therapeutic doses of MAOI-type B inhibitors (e.g. selegiline) will not adversely affect carbidopa–levodopa (25). Drugs that can increase the bioavailability of levodopa include antacids and metoclopramide. Metoclopramide, however, is a dopamine-blocking agent (121). Typical antipsychotic agents with high dopamine blockade may decrease the efficacy of levodopa (122). Pyridoxine (vitamin B6 )

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can increase peripheral conversion of levodopa; however, adding carbidopa to levodopa prevents this. Tricyclic antidepressants, methionine, hydantoins, benzodiazepines, and anticholinergics may decrease the efficacy of levodopa. Iron salts decrease the effectiveness of levodopa, and should be separated by at least 2 h. A protein-rich diet can interfere with levodopa absorption; regular carbidopa–levodopa may be taken at least 30 min before eating or 1 h after meals (123). Furthermore, rational, integrative management of PD requires: (1) dietary revision, especially to lower calories; (2) rebalancing of essential fatty acid intake away from proinflammatory and toward anti-inflammatory prostaglandins; (3) aggressive repletion of glutathione and other nutrient antioxidants and cofactors; (4) energy nutrients acetyl L-carnitine, Coenzyme Q-10, NADH, and the membrane phospholipid phosphatidylserine (PS); (5) chelation as necessary for heavy metals; and (6) liver P450 detoxification support. Psychosis in PD is a risk factor for nursing home placement and, in association with dementia, increases morbidity and mortality. Prior to the advent of atypical antipsychotic agents, attempts to treat psychotic symptoms led to inevitable worsening of motor function. Despite some encouraging advances in treatment strategies, our understanding of psychosis in PD remains in its infancy. The etiological basis of psychosis in PD remains an unsettled issue, although there is some controversial evidence that psychosis was observed in PD patients before the advent of levodopa therapy (123). However, the precise relationship between dopaminergic medications and psychosis has not been clearly delineated. The dose and duration of dopaminergic therapy are not considered consistent risk factors for hallucinations. The mechanisms that underlie PD psychosis remain to be determined. The most frequently proposed hypothesis is that, in some patients, denervation hypersensitivity of mesolimbic and mesocortical dopamine receptors may occur and that dopaminergic medications may stimulate these receptors to cause psychosis (123). More recently, it has been hypothesized that a combination or combinations of neurotransmitter systems play a role in the development of PD psychosis. It was suggested (124) that a serotonergic/dopaminergic imbalance may be most important. In support of this notion are postmortem studies revealing variable degrees and distributions of serotonin loss among patients with PD and reports that pharmacological agents that exert their effects on serotonin receptors (e.g. atypical antipsychotics, ondansetron) appear to have some efficacy against PD-related visual hallucinations (125,126). There appears to be a strong relationship between sleep disturbances and psychosis in PD. It continues to be debated whether sleep disturbances are a risk factor for psychosis in PD or if they represent one end of the “psychosis spectrum” in PD (126). In summary, psychosis in PD appears to be a drug-induced phenomenon. Antiparkinsonian medications alone, however, are not sufficient to cause psychosis (given the fact that only some medicated PD patients develop psychosis). There are likely to be neuropathological and pathophysiological differences among patients that result in the development of psychosis in some but not others. Supersensitivity of mesolimbic and mesocortical dopaminergic systems are likely to be involved, but nondopaminergic systems (and serotonin in particular) are probably important as well. Further elucidation of the close relationships between psychosis, and what are currently viewed as its biggest risk factors (dementia and sleep disorders), may provide insights into underlying mechanisms.

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For several reasons, however, there is a renewed interest in the neurosurgical community about the surgical treatment of PD. Firstly, it became evident that pharmacological therapy, which is the current mainstay of the management of PD, is usually unsatisfactory in the long term. As the disease progresses, the efficacy of that treatment often decreases, and incapacitating bradykinesia, rigidity, tremor, and impairment of gait and balance are frequently observed. Furthermore, late-course deterioration is frequently associated with debilitating levodopa-induced dyskinesias and fluctuations in clinical response (127). The implantation of cells genetically modified to express trophic factors and tyrosine hydroxylase for the synthesis of l-DOPA from tyrosine has been proposed as a possible route for the treatment of PD. Already, implantation of genetically modified cells that can secrete dopamine as well as l-DOPA has achieved a long-term correction of PD in rat models. If this technique is successfully applied to humans, it will probably be the treatment of choice in all parkinsonian patients in the near future. Coexistence of neurotransmitters with neuropeptides The progress in transmitter physiology has been of fundamental importance to all fields of medical science. During the last two decades, the number of putative neurotransmitters in the CNS has increased considerably and now comprises substances with large chemical differences, i.e. amines, peptides, purine, amino acids, prostaglandins, and leukotrienes. Until recent year, monoamines, acetylcholine, and amino acids were thought to be the only classical neurotransmitters. However, a large number of peptides, many of which were originally characterized in nonneural tissues, have been shown to exist also in CNS (128–130). These compounds are often referred to as neuropeptides and represent a heterogeneous group of molecules, the smallest ones built up of only two amino acids, with larger polypeptides consisting of 40 or more amino acids. Neuropeptides differ from the so-called classical transmitters since (1) they function in lower concentrations than classical transmitters, (2) synthesis of peptides is directed by mRNA in perikaryon as part of a much larger prohormone, from which the active peptide is cleaved by peptidase, whereas classical transmitters are formed from dietary sources by enzymes, (3) classical transmitters respond and adopt very rapidly to external stimuli and have their synthesis machinery with great capacity, whereas neuropeptides respond slower and their synthesis machinery have a more limited capacity and speed, and (4) molecular heterogeneity of peptides can exist, the different forms have different and sometimes opposite effects (131). Neuropeptides are widely distributed throughout the brain in specific nerve cells in coexistence with monoamines and other neuropeptides (131–134). They are believed to participate in several physiological and pathophysiological processes, including pain sensation, memory, neuroendocrine functions, regulation of release of monoamine transmitters, and regulation of mood (135). The effects are brought about by primary actions of neuropeptides or their modulation of the effects of monoamines transmitters. Transmission of nerve impulses from one neuron to another or from a neuron to a peripheral effector cell such as muscle cells is a critical event in the nervous system. Its elucidation is essential not only for our understanding of normal neuronal functions, but it is highly probable that changes in the chemical transmission process may underlie or

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at least are related to various disease processes in the nervous system. Moreover, it is now clear that many of the common drugs used to treat various diseases in the nervous system act via interfering with the chemical transmission process (136). Little more than 20 years ago, it became evident that a number of endocrine cells in the body are able to synthesize and secrete more than one hormone in each cell (135). These studies have also revealed that peptides in most instances are present in neurons that already contained, for example, catecholamines or the cholinergic transmitter acetylcholine or an amino acid transmitter. Several peptides, such as neuropeptide Y (NPY), cholecystokinin (CCK), substance P (SP), somatostatin, and vasopressin, are known to have ability to act as neurotransmitters. These peptides are also known to colocalize with classical neurotransmitters within a single neuron that provide a means to transmit more complex types of signals (136–138). Studies on the relationship between low-molecularweight transmitters and peptides in the CNS suggest the existence of both presynaptic and postsynaptic interaction between coexisting transmitters, in vivo and in vitro (139). One of the first examples of coexistence in the CNS was the demonstration of CCK-L1 in dopamine neurons in the ventral mesencephalon, especially in the ventral tegmental area (129). On the basis of coexistence with monoamine transmitters and distinct regional distribution, the assumption has been made that neuropeptides play a role in CNS disorders (140–145). The presence of receptor-active opioid peptides in human CSF was demonstrated more than 20 years ago (146). One of the major reasons for analyzing CSF peptides instead of plasma and serum is that neuropeptides do not readily cross the BBB, and CSF, due to constant exchange with the nervous tissue, rather than plasma, can be anticipated to contain peptides that derive from the CNS (143,144). The changes in the CSF peptide concentration might be attributed to certain symptoms and can even be interpreted as characteristic features of the pathological conditions (147). Neuropeptide Y, cholecystokinin, and SP are some of the important peptides that play vital biological roles in various degenerative disorders. The possibility of an additional type of interaction between dopamine and neuropeptides is suggested by recent findings which indicate that dopamine, like other classical neurotransmitters, is often colocalized in single neurons with neuropeptides and nontransmitter proteins (128,129). For example, certain midbrain dopamine neurons contain the peptide cholecystokinin (CCK), whereas other subpopulations of mesencephalic dopamine neurons contain the peptide neurotensin, and a third group of dopamine neurons in the ventral tegmental area contains CCK, neurotensin, and dopamine. Similarly, dopamine is colocalized in certain mesencephalic neurons with nontransmitter proteins, including acetylcholinesterase, protein-O-carboxymethyltransferase, cytochrome P-450 reductase, and a vitamin-D-dependent calcium-binding protein. The functional significance of such colocalization is not clear. However, because receptors for certain colocalized peptides such as neurotensin are present on dopamine neurons in the midbrain, activity of these dopamine neurons may be regulated by peptide autoreceptors in a fashion analogous to somatodendritic dopamine autoreceptor modulation of impulse flow and dopamine synthesis. Furthermore, because it appears that release mechanisms for dopamine and colocalized peptides may be dissociated under certain conditions (e.g. dependent on the firing pattern and firing frequency of the neuron, colocalized peptides may serve as part of a hierarchical array of neuronal regulatory features). In this regard, it is of interest that nerve

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terminal autoreceptors in the prefrontal cortex have been shown to exert reciprocal effects on dopamine and neurotensin release. Stimulation of dopamine autoreceptors diminishes dopamine release and enhances neurotensin release, whereas blockade of dopamine receptors augments dopamine release and diminishes neurotensin release. The functional implications of these findings for the activity of follower cells in the prefrontal cortex is presently uncertain, but it could allow the prefrontal cortex dopamine neurons to differentially modulate the physiological activity of cortical postsynaptic follower cells. Although the functional correlates of peptide–amine colocalization in mesencephalic dopamine neurons remains to be clearly established, it appears likely that colocalization of peptides or nontransmitter proteins and dopamine will prove to reliably define certain subpopulations of dopamine neurons. Thus, CCK-dopamine colocalized neurons of the ventral tegmental area project to the caudal, but not rostral, nucleus accumbens. Such distinctions may have important implications for regionally specific functions of dopamine in psychiatric and neurological disorders, as well as for the response of specific dopamine systems in these pathological conditions or in pharmacological treatment. The coexistence with neurotensin, particularly mesencephalic dopamine neurons, has not been observed in primates. However, this might not be a permanent phenotype (148). The possibility of a transient coexpression either in pathological states like schizophrenia or during ontogeny, similar to the transient multi-colocalization of tyrosine hydroxylase with peptides observed in the rodent amygdala (somatostatin and SP), seems plausible. In fact, some authors have recently found that the CCK gene is expressed in the midbrain of humans and, specifically, in the substantia nigra of schizophrenic patients, whereas CCK mRNA is low or nondetectable in the mesencephalon of normal subjects. Although neurotensin and CCK appear to modulate the function of mesotelencephalic dopamine neurons, their role in normal brain function or their possible dysregulation in neurological or psychiatric disorders or in stress- or drug-induced sensitization is still unclear. However, the availability of potent, bioavailable antagonists should lead to new insights concerning their importance and help to elucidate the role played by these neuropeptides in both normal and abnormal brain function (149–151).

NPY and its coexistence with noradrenaline Neuropeptide Y (NPY) is one of the most abundant and widely distributed neuropeptides in the mammalian CNS. NPY is a peptide consisting of 36 amino acid residues and has been shown to modulate a number of functions of CNS (152). NPY has several physiological effects (153), of which the most potent are an increase in vasoconstriction (154) and an increase in food intake. NPY is described as the most potent appetite stimulator known (153). It is found in high concentrations in several regions of the brain including nuclei of brain stem, and nerve fibers surrounding cerebral vessels has been proposed to play a role in regulating cerebral blood flow (CBF) and systemic vegetative functions. Vascular inflammation resulting in spasm and thrombosis has been documented during meningitis by histopathology and angiography and is likely to lead to focal education on CBF (152). Clinical and experimental studies have further documented that autoregulation of CBF is lost during meningitis, making CBF directly dependent on cerebral

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Controls PD (On) PD (Off)

200 180

NA

160 140 120 100 80

20

30

40

50

60

70

NPY

Fig. 5. Correlation between norepinephrine (NA) and NPY in CSF of PD patients (On and Off status) and healthy subjects.

perfusion pressure (155). Both occurrence of vasospasm and the loss autoregulation suggest that the regulation of cerebral vascular tone may be disturbed during meningitis. NPY plays an important role in anxiety, depression, and eating disorders. The coexistence of NPY with other neurotransmitters and its wide distribution in several brain areas predict the high importance of NPY as a neuromodulator. Thus, the effect of NPY on the release of several neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA), norepinephrine (NE), dopamine, somatostatin (SOM), serotonin (5-HT), nitric oxide (NO), growth hormone (GH), and corticotropin releasing factor (CRF) was recently reviewed (150). It has also demonstrated its coexistence with NA (151). Depression is a multifactorial process (156). Therefore, it is of interest to study the coexistence of NPY with NA, 5-HT, and DA systems irrelevant to depression since various neurological patients such as MS and PD commonly show signs of depression. The changes in NPY levels have been observed in different pathological conditions such as brain ischemia and neurodegenerative diseases (Huntington’s, Alzheimer’s, and Parkinson’s diseases). Taken together, these studies suggest that NPY and NPY receptors may represent pharmacological targets in different pathophysiological conditions in the CNS (157–161). Figure 5 shows the correlation between NPY and NA in both On and Off PD patients. Low levels of NPY in CSF has been reported in depressed patients, particularly those suffering from anxiety. All our PD patients showed increased CSF level of NPY. The correlation between the levels of NPY and NA shows a linear relationship for PD patients, which is similar to healthy controls. Cholecystokinin and its coexistence with catecholamines The discovery of the hormone that would eventually be called CCK dates back to the beginning of the twentieth century (162) and culminated in the sequencing of CCK-33

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by Mutt and Jorpes (163) in 1960. Subsequently, CCK-gastrin-like immunoreactive (CCK-L1) material was identified in the brain (164). Further studies confirmed that the octapeptide (CCK-8) is the main molecular variant of CCK in brain, and have also shown that it possesses biological activity comparable to synthetic sulfated (CCK-8s) (165). CCK plays a role in physiological, neurological, and psychiatric processes. Some major roles are gut motility, pancreatic secretion, and gall bladder contraction. In addition, CCK has effects on locomotor activity (166) and various reward-relevant behaviors, including cocaine and amphetamine self-administration (167) and food-related reward (168). Furthermore, CCK plays a role in schizophrenia, anxiety, and PD (169–171), and these findings have confirmed the neurotransmitter role of CCK. Our knowledge of CCK was greatly enhanced by the recent cloning of cDNAs of rat preprocholecystokinin and CCK receptor (172). Radioimmunoassay studies have revealed that CCK-L1 is found in high concentrations in the upper small intestine of the gastrointestinal tract, mainly localized in the duodenum and jejunum (173). In addition, it is also found in enteric nerves, ascending afferent fibers of vagus nerve, and even in the testis (174,175). Besides, CKK peptides attract considerable interest in research due to their part in termination of food intake, dopamine-regulated functions, and pain mechanism (176,177). While some of these effects are mediated by the CCK octapeptide sulfate (CCK-8s), other CCK peptides have different effects. CCK-8 is believed to be the most prevalent form of CCK in the CNS. It appears that CCK-8s and various analogs may modulate CNS dopamine neurotransmission and have been studied as potential antipsychotic agents (168). Among the other CCK peptides, its tetrapeptide (CCK-4) is anxiogenic in humans, and considerable amount of data supports its role in anxiety, with panicogenic properties and minimal gastrointestinal effects (177). Since CCK-ergic neurotransmission can be detected throughout the brain, it is not surprising that interaction occurs with many other neurotransmitter systems and that CCK has a variety of functional implications. Interactions with dopamine and with several other neurotransmitters such as GABA, serotonin, and noradrenaline are also of interest with regard to anxiogenic-like effects, its involvement in cognitive processes, the CCK-ergic modulation of opioid actions, and pain perception. Other functional roles for central CCK (octa peptide, CCK-8s) are as a mediator of satiety responses and as a regulator of sexual and maternal behavior and seizure activity. As may be expected from the involvement of CCK in several of these processes, CCK has also been shown to interact with other peptides and steroid hormones, and CCK apparently has a role as a modulator of learning and memory. The involvement of CCK (tetrapeptide, CCK) in anxiety responses is supported by numerous reports based on animal studies, as well as by clinical observation (173). Previously, we have shown (177) decreased levels of CCK-4 and CCK-8s in MS patients; however, the results in patients with PD, mood disorder, and eating disorders have indicated inconsistent observations (166). Our previous study (130) showed decreased level of CCK-4 in MS patients, whereas increased level in meningitis patients. The level of CCK-8s showed decreased tendency in PD patients. Figures 6 and 7 show a linear relationship between CSF CCK-8s and DA (Fig. 6) in healthy subjects, closely followed by PD patients, and a similar relationship is seen between CSF CCK-4 and NA in all PD patients (Fig. 7). This relation between these two values does show a linearity, indicating the dependence of these two transmitters on each other.

Neurotransmission, Oxidative Stress, and Coexistence of Neurotransmitters in PD 100

421

Controls PD (On) PD (Off)

80

DA

60

40 20 0 0

10

20 CCK-8

30

Fig. 6. Correlation between dopamine and CCK-8s in CSF of PD patients (On and Off status) and healthy subjects.

Controls PD (On) PD (Off)

240 220 200

NA

180 160 140 120 100 80 1

2

3

4 CCK-4

5

6

7

Fig. 7. Correlation between norepinephrine (NA) and CCK-4 in CSF of PD patients (On and Off status) and healthy subjects.

Substance P (SP) and its coexistence with dopamine and serotonin Substance P (SP) is an undecapeptide that derives from alpha, beta, and gamma preprotachykinin gene transcripts and is a neurotransmitter or neuromodulator of primary nociceptive afferents (178). SP is a naturally occurring tachykinin peptide isolated from brain tissues and the gastrointestinal tract. In the brain, substantia nigra and basal ganglia contain relatively high amounts of SP. There is evidence suggesting that SP functions as a neurotransmitter. It has been implicated in the pathophysiology of several

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neuropsychiatric disorders. SP may also serve as a useful tool in studying the effects of antidepressant drugs and electroconvulsive therapy (179). However, the contribution of SP to the understanding of neuropsychiatric disorders is far from clear. The possible functional significance of the coexistence of CCK and SP in neurons projecting to the spinal cord was tested by coadministration of the two peptides. At the doses tested, no synergistic interaction on the reflex was found with CCK and SP. IT MO caused a brief enhancement followed by a prolonged depression of the reflex. A high dose of CCK injected prior to MO increased the facilitatory effect and decreased the depressive effect of the opiate on the reflex. The effect of desulfated (D) CCK was similar to CCK, but at a higher dose. Naloxone (NAL) had a similar effect as CCK when administered prior to MO. The MO-induced depression of the reflex was readily reversed by NAL, but not by CCK. The results indicate that CCK may prevent the inhibitory effect of MO on spinal cord excitability to nociceptive stimulation, but does not reverse it. CCK may alter the balance of excitation–inhibition between the various types of dorsal horn interneurons that are involved in the transmission of nociceptive information (180). SP has received great attention due to its interaction with classical transmitters, especially with 5-HT, NA, and DA. In the CNS, SP is found in most regions with the highest levels in the substantia nigra and in dorsal horns of the spinal cord (181). SP is an undecapeptide with an almost established role as neurotransmitter or neuromodulator (172). The interaction between SP and serotonin is of importance in view of many studies linking serotonin to depressive illness (136). In pathways descending from the raphe nuclei, SP is found in the same neurons as serotonin (130). In accordance with the observation in rat, the basal ganglia are the regions in human brain most abundantly containing SP-L1, with the substantia nigra having the highest level. Very few studies are directed to compare the levels of SP in neurodegenerative diseases. However, in Huntington’s disease, patients showed (181) that SP-L1 levels in caudate nucleus and putamen are unchanged, while in globus pallidus and substantia nigra they are substantially reduced. Brains from patients suffering from PD also seem to have decreased levels of SP in globus pallidus and substantia nigra as compared to healthy controls (182). Unilateral injection of SP into substantia nigra of the rat evolves dose-dependent contralateral rotational behavior, possibly indicating activation of the ipsilateral nigrostriatal dopamine pathway (183). Furthermore, the behavioral excitation induced by a bilateral injection of SP into substantia nigra is abolished in rats with 6-OH DA lesion of the nigrostriatal dopamine pathway, suggesting a possible excitatory effect of SP on DA neurons. Very few studies are conducted on the levels of SP in CSF from the patients with neurological disorders (184,185). However, since SP plays a very important role in the CNS, it is very likely that CSF would provide us with its role in various neurological disorders. Our previous results (130) showed an increased CSF level of SP in CVD, TBM, and AM patients and a decreased level in MS patients. Figure 7 shows that a linear relationship is found in CSF between SP and 5-HT for healthy subjects as well as in PD patient groups, and that, irrespective of the different On or Off status, this relationship and its linearity are intact (Fig. 8). A similar relationship is seen between CSF DA and SP (Fig. 9). One possible mechanism may be the difference in the frequency dependence of the various transmitter pools.

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Controls PD (On)

70

PD (Off)

5-HT

60 50 40 30 20 2

3

4

5

Substance P

Fig. 8. Correlation between serotonin (5-HT) and Substance P in CSF of PD patients (On and Off status) and healthy subjects.

70

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DA

60

PD (Off)

50 40 30 20 3

4

5

Substance P

Fig. 9. Correlation between dopamine (DA) and Substance P in CSF of PD patients (On and Off status) and healthy subjects.

The release of classical transmitter with low molecular weight is generally believed to depend on the frequency by which action potentials invade the nerve terminal. Various studies (185,186) have shown that the release of neuropeptide as calculated per pulse recurred at high frequencies, compared to what is usually needed to evoke the release of classical small-molecule transmitters. In addition, neuropeptides were preferentially released by intermittent periods of stimulation at high frequency, compared to continuous stimulation. However, it is quite clear that despite the various characteristics of these

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neurological disorders, the relationship showing linearity between DA and 5-HT with SP remained intact. An interesting perspective on amine/peptide cotransmission has emerged due to the finding that chronic treatment with tricyclic antidepressant drug results in a marked increase in the tissue levels and release of SP from rat ventral spinal cord (186), suggesting that the mode of action of antidepressant drugs may be related to the changes in neuropeptide transmission.

CONCLUSIONS Neurodegeneration is the main consequence of PD, which is multifactorial, and there seems to be a cycle of steps involved. From these results, one can conclude that the neurodegeneration in PD patients involves free radical (oxidative stress) + cytostatic Ca2+ + mitochondrial damage + excitotoxicity of EAA and homocysteine + deficiency of vitamin B12 , B6 , and folate + role of transition metals, especially iron and copper. All these factors, one way or the other, result in the cell death process. Since our endogenous antioxidant defenses are not always completely effective, and exposure to damaging environmental factors is increasing, it seems reasonable to propose that exogenous antioxidants could be very effective in diminishing the cumulative effects of oxidative damage. Antioxidants of widely varying chemical structures have been investigated as potential therapeutic agents. However, the therapeutic use of most of these compounds is limited, since they do not cross the BBB. Therefore, any novel antioxidant molecule designed for potential neuroprotective treatment in acute or chronic neurological disorders should have the mandatory prerequisite that they can cross the BBB after systemic administration. Neuroprotection is a key issue in the modern management of PD. However, none of the currently available antiparkinsonian treatments have proven to retard disease progression and to provide a neuroprotective effect. It is concluded that the future of therapy in PD is likely to include a “cocktail” of neuroprotective compounds to interfere with several molecular pathways that lead to neuronal injury. In using therapeutic strategies aimed towards retarding or arresting neuronal death, close attention will need to be paid to quality-of-life issues. One of the main problems during the many years of research on PD has been to detect the disease early. New methods for early detection and for monitoring disease progression to help the clinicians in their clinical diagnosis are important, particularly in future pharmacological treatments that can halt further neurodegeneration and nerve cell death. One of the tools that have been used during last few decades is to examine the patient’s brain with positron emission tomography (PET). Combining this technique with analysis of biochemical, pathological, and behavioral changes, more information on PD patients can be monitored. On the basis of our study, the results on degenerative disorder, the roles of various neurotransmitters, and the roles of free radical NO, cobalamin, and homocysteine are clearly defined in order to develop effective drug therapy. This may include glutamate-releasing inhibitors, excitatory amino acid antagonist agents to improve mitochondrial function, free radical scavengers, and neuroprotective agents as antioxidants along with vitamin B12 . Mortality remains

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abnormally high in PD, and improving life expectancy is the major objective for future antiparkinsonian treatment.

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