Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.294542
Source of Support: None, Conflict of Interest: None
Keywords: Benomyl, nucleus pars medialis, optic tectum, Parkinson's disease, zebrafish
Parkinson's disease (PD) is defined as a progressive and chronic neurodegenerative disorder that is prevalent worldwide in 31 to 328 per one lakh people. It results due to the progressive and idiopathic degeneration of dopamine-producing neurons in the substantia nigra (SN) region of the midbrain. Resting tremors, cogwheel rigidity, and bradykinesia are the three “cardinal signs” with postural instability as the late finding in PD. PD is caused by the pathologic alterations in the basal ganglia of the cerebral hemisphere which is called the extrapyramidal system. A complex circuitry of afferent and efferent neurotransmitter pathways connects regions of the basal ganglia with each other and to other brain areas. Dopamine is the major neurotransmitter within the extrapyramidal system; 80% of total brain dopamine occurs in the corpus striatum. The widespread distribution of dopamine neurons proves that PD may be system-wide rather than occurring as lesions. This shows that dopamine may exert both an excitatory and/or an inhibitory property that results in a more generalized modulating influence on nigrostriatal neurons than controlling the neuronal discharge. Acetylcholine (Ach) balances the release and activity of dopamine within the extrapyramidal system; hence, the movement disorders hold an important characteristic symptom in PD.
The site of pathologic involvement in PD is the SN with dopamine neuronal degeneration as well as dopaminergic neurotransmission. In a healthy person, dopamine neurons in SN (appears as grey matter) release sufficient dopamine to control the stimulating effect of acetylcholine on the large motor and fine muscle movements. As a result of the progressive damage to the nigrostriatal dopaminergic neurons in PD, an insufficient amount of dopamine (DA) is generated as a counterbalance to Ach production leading to excessive motor nerve stimulation. In addition, levels of homovanillic acid (HVA) and enzymes such as dopa decarboxylase are reduced in PD patients. Lewy bodies (LBs) are also the notable feature of PD that affects the substantia nigra pars compacta and other brain areas. Hyper expression of α-synuclein in neurons lead to its accumulation in the ER and caspase-dependent nonapoptotic cell death; further triggers the unfolded protein response (UPR) due to ER stress which leads to the release of cytochrome C from the mitochondria and thus elevates the dendritic mitochondrial oxidant stress in dopaminergic neurons., Certain pesticides, namely, paraquat, hepatochlor, atrazine, and chemicals such as MPTP inhibit NADH in the mitochondrial electron transport system (ETS). Familial PD is also caused by mutations in PINK-1, a mitochondrial kinase involved in mitochondrial fission and mitophagy., Oxidative damage in mitochondria leads to deficiencies in the complex I, II, and III which further leads to raised free radical production and reduced “ubiquinone”. Intracellular rise in calcium is also manifested in PD due to mitochondrial damage which further promotes the α-synuclein aggregation.
Management of PD
L-dopa is the mainstay drug and of primary choice in PD that has limited usage as it develops motor fluctuations and dyskinesias. L-dopa can be combined with dopamine agonists as they mimic the endogenous dopamine release from the dopamine receptors. Monoamine oxidase B inhibitors like selegiline contribute to the rise of dopamine levels in the basal ganglia by inhibiting dopamine catabolism. Catechol O-methyl transferase (COMT) inhibitors also inhibit catabolism of DA and extend the peripheral half-life of L-dopa. Treatment of PD in early stages diminishes the symptoms and helps to maintain independent functions; whereas in the advanced stages, the treatments work on maximizing “on” time i.e., the duration up to which the drug is effective and minimizing the dyskinesia, motor fluctuations, and psychiatric problems. In patients with chronic PD and intolerable side effects, surgical methods such as deep brain stimulation, pallidotomy, and tissue transplantation were employed.
Alternative models in Parkinson's disease and their limitations
Parkinson's disease does not develop naturally in animals except in humans. In experimentation, dogs, cats, monkeys, rats, and mice were commonly employed by inducing neurotoxins like MPTP, 6-hydroxydopamine (6-OHDA), paraquat, rotenone, and so on but their utility was limited by considering the costs and ethical concerns Almost all these models leads to dopamine depletion, mitochondrial dysfunction, formation of reactive oxygen species and hence are successively meant to study the mechanism, onset, and progression of the disease as well as the therapeutic efficacies but still could not pronounce the exact clinical and pathological aspects as in human PD.,
Different models such as yeast, worms, and fruit flies are used to investigate the fundamental cellular processes such as apoptosis, autophagy, oxidative stress, protein functions, and so on involved with PD thereby studying the expression of human genes in these organisms. For a decade; teleost fish, especially the zebrafish has nailed a worthwhile role to study the neurodegenerative pathways. Almost all the neurotoxins were employed in the teleost fish to enlighten the hidden pathological aspects in PD. This review particularly explains the shrewdness of goldfish and zebrafish in PD.
Zebra fish and Parkinson's disease
Danio rerio, commonly known as zebrafish (order: cypriniformes; class: Actinopterygii), established in south Asia has transpired as an excellent model organism for studies on vertebrate biology. The male and female zebrafish are easily distinguishable; males are torpedo-shaped with gold and blue stripes, while females have a larger, whitish belly with silver stripes. This makes the breeding easy to obtain a large number of progenies. The high fecundity of the female zebrafish makes it an excellent model for genetic screens and analysis. Moreover, zebrafish can tolerate temperature fluctuations, lighting, and a reasonable amount of stress. The breeding can be timed, easily controlled by changing the light-dark cycle and a large number of embryos from a single batch of fish (large progeny) laid significance in the utilization of zebrafish for any kind of experiment. Their transparent larvae facilitate localization of proteins, genetic, and pathological manipulations. Zebrafish activity has proven a viable endpoint for detecting neurological impairments received during development and hence gained a lot of importance in studying neurodegenerative disorders. The occurrence of specific brain regions comparable to human counterparts, the dopaminergic system in the zebrafish brain interacts extensively with serotoninergic and histaminergic systems and simplicity in the genetics of zebrafish, made it a good model to study the genetics of PD. Orthologs for genes such as pink1, parkin, lrrk2, and dj-1 responsible for Mendelian PD have been found in zebrafish and studied by implementing methodologies such as MO knockdown or transgenic overexpression of mutants. Neurotoxins MPTP, 6-OHDA, and pesticides effectively produce Parkinson like symptoms in zebrafish leading to dopamine deficiency and dopaminergic neurodegeneration.
1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) model in zebrafish
Anichtchik et al. in 2014 utilized zebrafish for the first time in Parkinson's study by inducing MPTP as well as 6-OHDA individually. In this study, adult zebrafish were divided into 7 groups; one was the control group (0.9% NaCl) and 2–7 other groups were injected with different doses of MPTP hydrochloride (20, 40, and 80 mg/kg, i.p) and 6-OHDA hydrobromide (25, 50, and 100 mg/kg, i.m) using Hamilton syringes. Based on the pilot studies, MPTP treated zebrafish groups were observed on 1, 2, 5, 7, and 12 days for behavioral changes and HPLC measurements of catecholamine levels; whereas, the 6-OHDA groups were assessed on 1, 3, 6, and 9 days after injection.
The locomotor activity of the fish was observed using a video tracking system. The total distance moved, the mean velocity that characterizes the general locomotor activity, mean turning angle, and place preferences in the tank were recorded. A marked decrease in the general locomotor activity altered complexity of the swimming path was well correlated with the levels of brain catecholamines such as dopamine and noradrenaline concentrations which were analyzed using HPLC associated with an electrochemical detector.
The zebrafish nervous system advances within 72 h post-fertilization; whereas, the dopaminergic system develops in 18 h. Lockwood et al., 2004 and Orger et al., 2004 have evaluated larval behavior in light using optical tracking devices., Infrared image analysis and the locomotor activity in both light and darkness were also studied by Emran et al. 2007 and Prober et al. 2006., The larvae (6 days post-fertilization) were maintained in 96-well microtiter-plate wells in 10% Hanks' solution. MPTP like neurotoxins and test compounds were administered to the larva in the well plates. The larval fish movement was tracked using a video camera and parameters such as the distance of the larval movement, larval activity pattern in light and dark conditions, etc., The activity of larvae was initially high and then decreased in a dark compartment, whereas, it has a subsequent increment in the case of the light compartment which indicates that the light period played an important role in enhancing behavioral activity. It was also evident in all the above studies that neurotoxins play a lead role in depriving the activity of larval fish as well as adult fish.
Pesticides, Parkinson's disease, and zebrafish
Barbeau and his coworkers in 1987 have explained the association of pesticides with PD. Prolonged exposure to certain pesticides like benomyl at the workplace is prone to PD. The pathology involves inhibition in the electron transport chain leading to mitochondrial dysfunction (similar to MPTP action), oxidative stress ad impairment in the ubiquitin-proteasome process and mainly by inhibition of aldehyde dehydrogenase (ALDH), and an enzyme which plays a critical role in DOPAL detoxification. DA is converted to DOPAL (3,4- dihydroxy phenylacetaldehyde) which is highly toxic. Further by the action of enzyme aldehyde dehydrogenase, DOPAL is converted to a less toxic metabolic, DOPAC (3,4- dihydroxy phenylaceticacid); this step is inhibited by the pesticides. Hence, in thein vivo studies of pesticide-induced PD using zebrafish, the ALDH in liver and brain mitochondria was found to be depleted.
Casida et al. in 2015 have administered 40 mg/kg benomyl (i.p) in mice, isolated the brain samples to evaluate the catechol levels, and documented an increased level of DOPAL and decrease in DOPAC. ALDH depletion in the postmortem brain samples with no signs of Lewy body's formation of PD individuals was also documented.
Role of mutant zebrafish in Parkinson's disease
The rapid development and survival of the zebrafish embryo and larvae, studies of gene expression and function can be successfully carried throughout early developmental stages. In addition, the simplicity and effectiveness of manipulating gene expression in the embryos permit the researchers to study cell biology in relevance to human pathology. Several mutant genes were identified in zebrafish for PD. The zebrafish protein degylcase (DJ-1 protein), 80% homology with human and mouse gene is expressed throughout the body. The ubiquitin carboxy-terminal hydrolase L-1(UCH-L1) was detected in the ventral region of the midbrain, hindbrain, and in the ventral diencephalon.Parkin, ubiquitin processing gene (60% homologous with human Parkin) has gained much importance in Parkinson's studies; leads to a significant decrease in the number of dopaminergic neurons of the posterior tuberculum (homologous to the substantia nigra in humans).
The goldfish (Carassius auratus) is a common aquarium and freshwater fish belonging to the family Cyprinidae of order Cypriniformes. Goldfish is prone with associative learning skills and the visual acuity that helps to distinguish individual humans. The goldfish rapidly swims around the tank, moves to the surface to grab the food, and hides when other people approach the tank. It has a memory span of at least 3 months and can be trained to recognize and to react to light signals of different colorsor to perform tricks. This behavior of the goldfish laid a foundation for its role in neurodegenerative disorders.
The MPTP model in goldfish leads to the occurrence of PD with respect to the biochemical and histologic pattern engendered in the primate model. Usually, in lower vertebrates, MPTP could not elicit PD-like movement disorder as the structure similar to SN in the teleost midbrain is absent. The nervous system of the goldfish is simple and has a highly lipophilic blood-brain barrier that helps in the uptake of majority neurotrophic and neuroprotective compounds; accessible for the study of PD. Studies performed by Brodie et al. and Nagatsu et al. have reported the existence of serotonin, noradrenaline, and tyrosine hydroxylase in the forebrain and midbrain regions of goldfish. Further, Youdim and his coworkers have reported the occurrence of monoamino oxidases (MAO) A and B, DA, and noradrenaline (NA) in the fore, midbrain, and vagal lobes of goldfish. The presence of “telencephalic nucleus pars medialis” (NPM) in the forebrain (equivalent to the SN of humans) is a considerable property of goldfish to study Parkinsonism More Details that can be induced by a single i.p. administration of MPTP, similar to human PD with symptoms like bradykinesia, the appearance of 1-methyl-4-phenylpyridinium (MPP+), and the loss and recovery of both DA and norepinephrine (NE). Goldfish model is even advantageous than other Parkinson models as within 10–13 days, the fish can be recovered to the normal behavioral movements and normal brain monoamine levels occur within 10–13 days.
Pollard et al. (1997) showed that a single intraperitoneal administration of MPTP (50 mg/kg) leads to the accumulation of MPP+ in the brains of goldfish which further cause a sustained depletion in DA and NA levels. There is also a change in the behavioral parameters that were studied by assessing the “swimming movements/mobility” in an “activity meter”. Total distance traveled, time of rest, and the apparent rate of motion made by the fish within the 5-minute period will be computed by the activity meter. This study is to be performed for 24, 48, 72, 96, and 120 h after MPTP administration. Further observations like a decrease in the food intake by the MPTP treated fish are also made.
Tipton and Youdim designed a measure for the monoamine oxidase activity in the brain of goldfish. High-performance liquid chromatography with electrochemical detection is performed to study the DA, NA, and serotonin levels and amino acid concentrations were determined by high-performance liquid chromatography with fluorimetric detection. Darkening of cytoplasm and mitochondrial inflammation were also considered as measures for the depletion of DA and NA., In the retina of goldfish, as MPP+ gets collocated in the vesicular compartment of the neuron and could not to the cytoplasm and mitochondria, there occurs no damage to the retina. In addition, MPTP shows a marked reduction in the tyrosine hydroxylase (TH)-immunoreactive cell count in the ventral mid-telencephalon, and was blocked by the selective MAO-B inhibitor, L-deprenyl i.e., the damaging effects of MPTP are interfered by MAO-B in goldfish brain; this is similar as described for mammals and rodents. Moreover, the reduction of GABA-glutamate and glutamine acid decarboxylase (GAD), in the MPTP-treated goldfish was studied. In contrast, GAD67 and GABA synthesis were upregulated in the optic tectum and telencephalon of the MPTP treated brains of goldfish. The authors suggested a parallel between the goldfish MPTP model and the primate MPTP model, which includes the apparent similarity in DA modulation of GAD67.
Callard et al., in 2001 studied the relationship between estrogen and dopamine in the neurons of Parkinson's patients. Estrogen helps in neurodegeneration and protects the brain against apoptosis, injury, and ischemia through several mechanisms such as nuclear ER-mediated mechanism, membrane receptor-mediated actions, and free radical scavenging mechanism. Studies performed by D'Astous et al., and Shughrue showed that estrogens and estrogenic drugs protect existing nigrostriatal DA neuron activity including the DA concentration, TH expression, dopamine transporter (DAT) expression, and neurotrophin release and suggested that the implication of ERs rather than their antioxidant activities are involved in the effects of estrogens on DA neuron activity.,, Microarray results from previous studies showed that injecting the dopaminergic neurotoxin MPTP and dopamine receptor1 a (D1R) agonist (SKF 38393) can up-regulate or down-regulate cyp19a1b expression, respectively, which confirms that cyp19a1b expression can be regulated by neurotransmitter DA in goldfish. MPTP induction in female goldfish leads to DA neuron degeneration and aromatase inhibition successfully. This finding pronounces the involvement of estrogens as neuroprotective in the neurotrophic factor synthesis.
Comparison between goldfish and zebrafish
Although the above mentioned factors have made goldfish advantageous in research, still it has not been utilized as efficient as zebrafish because of its phylogenetic proximity to zebrafish., As several manipulation techniques were established in zebrafish, the large size of goldfish embryos has become insignificant which was once very advantageous in molecular biology and developmental genetic studies. The embryological similarities and phylogenic proximity of these two teleost species suggest that studies of goldfish would not discover novel molecular phenomena as it is already proved in zebrafish studies. But according to observations of Schilling et al. in 2007, almost no researchers have specifically indicated the potential of the goldfish morphological variation for evo-devo studies.
Komiyama in 2009 showed that Goldfish (Carassius auratus) has a unique property to survive in the complete absence of molecular oxygen for a long period and has twice the number of chromosomes (n = 50) than zebrafish (n = 25) and other teleosts. Therefore, this shows an additional round of whole-genome duplication (WGD) event which is considered as the fourth-round (4R); goldfish chromosomes showed 2:1 synteny to zebrafish chromosomes, providing evidence for the fourth round of WGD in goldfish of WGD which would be a valuable model to study the consequences of the genome. These characters prove that goldfish is a suitable model to study genome duplication and physiological adaptation genome evolution, physiology, and neurobiology studies.
This review provides an emphasis on the utilization of alternative models in neurodegenerative disorders like PD. Since the 1980 s, goldfish have been utilized other animal models and later were being replaced by zebrafish. Similar to animal models, zebrafish can also be studied effectively in terms of different induction models, genetic patterns, and mutations. Although alternative models have advantages there are still some pitfalls in assessing the exact outcomes. We can now conclude that zebrafish appears as a contemporary to the MPTP models of PD in primates, in terms of neurochemical, neuroanatomical changes, and movement dysfunctions. More sophisticated methods for analyzing the motor functions of goldfish should be developed and implemented.
Recent studies have suggested that environmental factors have a crucial role in triggering and/or propagating the pathological changes in PD. Although many studies have been and being performed by utilizing MPTP like chemicals to study the effectiveness of new extracts and compounds in PD, a little focus was made on the role of pesticides. Since agricultural fields account for 37.7% of land area worldwide and the use of pesticides is an important risk factor in neurodegeneration, there is a crucial need to focus on the association between pesticides and PD.
The authors are thankful to the Dean SRM College of Pharmacy and staff, department of Pharmacology for their great review and suggestions.
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Conflicts of interest
There are no conflicts of interest.