Screening modelS
for
ParkinSon’S diSeaSe
Arup Kumar Bishoyi
Roll No – PC/2017-X/166
Dept. Pharmacology &
Toxicology
NIPER- Guwahati

Content
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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,
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Dauer W et al. Neuron. 2003 Sep 11;39(6):889-909

Motor circuit in PD pathology
Cerebral cortex
Striatum
D2 receptor D1 receptor
GPi
GPe
STN
VA & VL
Thalamus
SNpc
+
- -
-
+
+
-
+
X
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Pathophysiology
Poewe, W. et al. Nat. Rev. Dis. Primers 3, 17013 (2017).
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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
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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
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PROCEDURE
• Animals: male NMRI mice (18–22 g)
• Std. drug -5 mg/kg benzatropine mesilate
• Inducer: 0.5 mg/kg oxotremorine s.c.
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Evaluation
• Hypothermia
• Tremor
• Lacrimation
• The model measures only central anticholinergic activity
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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
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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.
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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
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Cont…
ROS
BAX
Cyt-C
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Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003 Sep 11;39(6):889-909.

EVALUATION
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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
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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
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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
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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
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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
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Blanco-Ayala T et al. 2014 Jun 1;48(6):623-40.

Genes
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Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron. 2003 Sep 11;39(6):889-909

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.
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Koprich JB et al. Nature Reviews Neuroscience. 2017 Sep;18(9):515.

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
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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.
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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
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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.
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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.
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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
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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.
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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.
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