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  1. Screening modelS for ParkinSon’S diSeaSe Arup Kumar Bishoyi Roll No – PC/2017-X/166 Dept. Pharmacology & Toxicology NIPER- Guwahati
  2. Content 05/18/18 2
  3. Introduction  Parkinson disease is the second-most common progressive neurodegenerative disorder that affects 2–3% of the population ≥55 years of age.  In PD there is deficiency of dopamine in Nigrostriatal dopamine pathway.  It is important to remember that,only ∼10% of PD cases are due to genetic mutations  PD Characterized by cardinal features of resting tremor, rigidity, bradykinrsia, 05/18/18 3 Dauer W et al. Neuron. 2003 Sep 11;39(6):889-909
  4. Motor circuit in PD pathology Cerebral cortex Striatum D2 receptor D1 receptor GPi GPe STN VA & VL Thalamus SNpc + - - - + + - + X 05/18/18 4
  5. Pathophysiology Poewe, W. et al. Nat. Rev. Dis. Primers 3, 17013 (2017). 05/18/18 5
  6. Screening Models Tremorine & oxotremorine antagonism Reserpine antagonism model • MPTP model in monkeys • 6-OHDA induced model • Pesticide induced models Genetic models MitoPark mouse model Drosophila model C. Elegans model Zebrafish model 05/18/18 6
  7. Tremorine & oxotremorine antagonism • Purpose • Muscarinic agonists tremorine and oxotremorine induce parkinsonism-like signs like tremor, ataxia, spasticity, salivation, lacrimation and hypothermia • Antagonized by anticholinergic drugs 05/18/18 7
  8. PROCEDURE • Animals: male NMRI mice (18–22 g) • Std. drug -5 mg/kg benzatropine mesilate • Inducer: 0.5 mg/kg oxotremorine s.c. 05/18/18 8
  9. Evaluation • Hypothermia • Tremor • Lacrimation • The model measures only central anticholinergic activity 05/18/18 9
  10. Reserpine antagonism Purpose •Reserpine works by inhibiting the vesicular monoamine transporter, VMAT2. •Reserpine produces ~85% loss of dopamine in the SNpc. >95% dopamine depletion in the striatum (2 h inj) Procedure •Animals: Male NMRI mice (20–25 g) •Inducer: Reserpine ( 5 mg/kg i.p) •30 min prior to observation the test compounds are injected 05/18/18 10
  11. Cont… • The animals are placed singly onto the floor of a Perspex container • Horizontal movements are recorded for 10 min • Record rearings and grooming episodes. 05/18/18 11
  12. MPTP model in monkeys (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) •MPTP is indeed the gold standard for toxin-based animal models of PD. •MPTP primarily causes damage to the nigrostriatal DA pathway with a profound loss of DA in the striatum and SNpc PROCEDURE •Animals: rhesus monkeys( 5–8 kg) •Inducer: N-MPTP •Dose: 10– 18 mg/kg i.v •Time period : 5-8days 05/18/18 12
  13. Cont… ROS BAX Cyt-C 05/18/18 13 Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003 Sep 11;39(6):889-909.
  14. EVALUATION 05/18/18 14
  15. 6-OHDA Induced models • 6-OHDA does not efficiently cross the blood–brain barrier and so requires direct injection into the brain. 6-OHDA unilaterally injected by using stereotaxic surgical instruments 6-OHDA is taken up into the dopaminergic neurons via the dopamine transporter, DAT • 6-OHDA initiates degeneration through a combination of oxidative stress and mitochondrial respiratory dysfunction. • 6-OHDA readily oxidizes to form reactive oxygen species (ROS) such as H2O2 Dopaminergic neurodegeneration 05/18/18 15
  16. Circling behavior in nigrostriatal lesioned rats • Unilateral lesion of the dopaminergic nigrostriatal pathway in the rat by the neurotoxin 6-OHDA • Rat are rotates in a opposite direction (contralateral) when apomorphine, or L- dopa is given. • Rats rotate towards the lesioned side (ipsilateral) when amphetamine is administered • Rotational behaviour is dependent upon the amount of DA receptor stimulation, and the extent of DA denervation produced by the 6-OHDA lesion • This test is used for the study of central dopamine function and the evaluate the mode of action of new drugs on dopaminergic neuron 05/18/18 16
  17. Procedure • Animals: Male Wistar rats (200–250 g) • Anaesthesia: sodium pentobarbital(60 mg/kg i.p.) • Toxin: 6-OHDA(8 μg) Rats are anaesthetized with sodium pentobarbital. Unilateral lesion done by using stereotaxic instrument Test compounds are given i.p or sc. and the animals placed into the roto meter Circling is recorded over a 1 h period. For contralateral circling are determined by injecting apomorphine at 1mg/kg s.c. and recording the rat’s circling for 1 h Percent change of drug turns from control & test turns is recorded. Using various doses ED50 values can be calculated Determine the for ipsilateral turning, each subject is administered 2.5 mg/kg of D- amphetamine 05/18/18 17
  18. Rotenone model • Rotenone is the most intoxicating member of the rotenoid family and is typically found in tropical plants. • It is both an herbicide and insecticide having a half‐life of 3–5 days • The most commonly administred by systemic route using osmotic pumps in rats, (2–3 mg·kg-1/day) • Rotenone is known to be a high-affinity specific inhibitor of complex 1 ,that are involved in oxidative phosphorylation 05/18/18 18
  19. Paraquat & Maneb model (N,N dimethyl 4 4 bypiridinium) ‐ ‐ ‐ ‐ •paraquat (10 mg/kg i.p.) •Maneb (30 mg/kg i.p.) •Paraquat (PQ) is an herbicide that exhibits similar structure to MPP+ •Maneb preferentially act by inhibiting complex III of the mitochondrial respiratory chain. •PQ and Maneb have been shown to produce enhanced toxicity when combined • PQ exerts its deleterious effect through oxidative stress mediated by redox cycling and generating reactive oxygen species(ROS) } 6weeks 05/18/18 19 Blanco-Ayala T et al. 2014 Jun 1;48(6):623-40.
  20. Genes 05/18/18 20 Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003 Sep 11;39(6):889-909
  21. α-synuclein  α-synuclein expressed in the nervous system, where it is found in presynaptic nerve terminals  α-synuclein is a protein that, in humans, is encoded by the SNCA gene  Mutations of α syn, done by any of the substitutions A30P, A53T, and E46K ‐ encoding genes.  Mutations in a-synuclein increase the propensity for misfolding leads to formation of lewybodies. 05/18/18 21 Koprich JB et al. Nature Reviews Neuroscience. 2017 Sep;18(9):515.
  22. LRRK2 • The most frequent mutations are the G2019S and the R1441C • Overexpression of wild-type LRRK2 in BAC transgenic mice induces increased DA release in the striatum and motor hyperactivity • BAC transgenic mice overexpressing R1441G mutant LRRK2 protein showed age-dependent and progressive motor-activity deficits, • LRRK2 KO mice are viable and have an intact nigrostriatal DA pathway up to 2 years of age • Mutated LRRK2 prompts the aggregation of a-synuclein 05/18/18 22
  23. Parkin • Parkin is an E3 ubiquitin ligase and participates in the ubiquitin proteasome system. • Combination with PINK1, parkin is directly involved in the mitochondria quality control. DJ-1 • DJ-1 is a redox-sensitive molecular chaperones protein, localized in the cytoplasm, which associates with mitochondria. • DJ-1 KO mice are more sensitive to toxins and oxidative stress. • DJ-1 overexpression was associated with increased protection against toxin- induced neurodegeneration. 05/18/18 23
  24. MitoPark mouse model Mitochondrial function is selectively disrupted by elimination of the nuclear genome encoded mitochondrial Tfam gene Mice survive to adulthood and progressively show a PD like motor deficts Degeneration of nigrostriatal pathways. L-dopa treatment normalizes motor deficits of MitoPark mice and its efficacy weakens with age 05/18/18 24
  25. Drosophila model • fruit fly Drosophila melanogaster has emerged as a suitable model for studying mechanisms of PD-related neurodegeneration. • the models exhibit motor deficits, manifest as a premature loss of climbing ability when the flies are permitted to ‘escape’ from a vial housing them a-synuclein transgenic Drosophila model (A53T or A30P ) premature loss of climbing ability. degeneration of dopaminergic neurons. A-synuclein formation. PINK1 Knock out in Drosophila reduced numbers of Dopaminergic neurons &impaired climbing ability. Rotenone induced Parkinson’s Disease inhibition of climbing in 80% flies. 30–50% loss of TH +ve neurons. 05/18/18 25
  26. C. elegans • One-third of the cells that constitute the adult worm are neurones, of which exactly eight are dopaminergic and are involved in motor activity • Mutations within the gene leads to the typical decrease in locomotion (basal slowing) i.e. reduction of bending frequency when near food(e.g. bacteria), to facilitate feeding. • C. elegans is transparent and dopaminergic neurones can be easily observed in live animals by driving the expression of green fluorescent protein. • by over-expressing wild-type or mutant human a-synuclein (A53T or A30P) display degeneration of dopaminergic neurons alongside loss of the basal slowing response. • over-expression of wild-type LRRK2 increased survival in response to paraquat and rotenone indicating LRRK2 mutations may enhance vulnerability in PD. 05/18/18 26
  27. Zebrafish model • In zebrafish, dopaminergic neurons found in the posterior tuberculum of the ventral diencephalon ascend towards the striatum. • This altered dopaminergic activity is mirrored by reduced swimming, indicative of bradykinesia, • Overexpression of parkin protein protects the fish from cellular stress. • Parkin KO causes a moderate (20%) loss of dopaminergic neurones, reduced mitochondrial complex I activity and increased susceptibility to toxins • PINK1 knockdown in zebrafish does not induce dopaminergic cell loss but, instead, alters dopaminergic projections and induces locomotor deficits 05/18/18 27
  28. Summary • Pharmacological models are not effective for drugs used for repeated administration. • Toxin models are effective but they don’t show the formation of α-synuclein aggregrates & lac of age dependent degradation of dopaminergic neuron. • Genetic model of Parkinsons disease are effective but not showing the neurodegeneration which is one of major hallmark of PD patiennts. • Alternative models are more effective as compare to all model as they shows all the symptomps occurred in PD patient . • Selection of a suitable model which shows all the cardinal feature of PD is effective for new drug development of parkinsons disease. 05/18/18 28
  29. REFERENCES • Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, Schrag AE, Lang AE. Parkinson disease. Nature reviews Disease primers. 2017 Mar 23;3:17013. • Koprich JB, Kalia LV, Brotchie JM. Animal models of α-synucleinopathy for Parkinson disease drug development. Nature Reviews Neuroscience. 2017 Sep;18(9):515. • Blesa J, Trigo-Damas I, Quiroga-Varela A, del Rey NL. Parkinson’s Disease- Associated Mutations Affect Mitochondrial Function. InMitochondrial Mechanisms of Degeneration and Repair in Parkinson's Disease 2016 (pp. 139-158). Springer, Cham. • Duty S, Jenner P. Animal models of Parkinson's disease: a source of novel treatments and clues to the cause of the disease. British journal of pharmacology. 2011 Oct 1;164(4):1357-91. • Blesa J, Przedborski S. Parkinson’s disease: animal models and dopaminergic cell vulnerability. Frontiers in neuroanatomy. 2014 Dec 15;8:155. • Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003 Sep 11;39(6):889-909. 05/18/18 29
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