Nicotinic receptors and parkinsons disease

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• Nicotinic receptors and parkinsons disease
• By Maryka Quik and
• Gayathri Jeyarasasingam
• European Journal of Pharmacology 393 (2000)
  223-230
• Presented by Natasha Frett

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Introduction

• Parkinsons disease is a neurodegenerative
  disease that results from loss of dopaminergic
  neurons.
• Greatly effects dopaminergic neurons in the basal
  ganglia- substantia nigra.
• The neurons release dopamine.
• Sends inhibitory stimuli to the cerebral cortex.
• Excitatory stimuli (ACh) is sent by the
  cerebellum.
• The action of both causes smooth voluntary
  movement in muscles.

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• Nicotinic-acetylcholine (nAChR) receptors
• cholinergic ligand-gated cationic channels
• Most nAChRs are presynaptic neurons
• composed of several subunits randomly arranged
  to give receptors that are slightly different.
• subunits (?2-?7 and ?2-?4) form pentameric
  channels that are 300 kDa

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• Acetylcholine at dentritic portion of neuron is
  received by nAChR
• allows the influx of Ca 2
• Causes release of synaptic vessicles

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• Nicotine
• agonist
• decreases receptor breakdown
• inhibits monoamine oxidase B, the enzyme that
  breaks down dopamine

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• Stimulation of the nAChR receptors causes an
  influx in Na and Ca2.
• influx depolarizes the cell and regulates the
  release of neurotransmitters,
• modulates neurite outgrowth,
• Neurite out growth plays an important role in
  enhancing cognition and reducing dementia
• activation of secondary messengers
• Early gene transcription

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• The different combinations of subunits give rise
  to the different pharmacologic properties.
• ?7 subunits have a high permeability to Ca2
• Combinations of subunits affect the rate of
  desensitization.
• ?3?2 desensitizes 10 times faster than ?3?4 or
  ?4?4

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Protective subunits

• ?7 has neuro-protective against glutamate
  toxicity, adverse effects of nerve growth factor
  deprivation and ethanol induced cytotoxicity.
• Activation of ?4?2 containing receptors protects
  against ?-amyloid-induced toxicity

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• Objective To
• Observe the distribution of mRNA subunits in the
  substantia nigra in a monkey model after inducing
  Parkinsons using MPTP.
• Compare this mRNA distribution to that of the
  mesencephalic (midbrain) rodent model.
• Observe the neuro-protective effect of nicotine
  in a Parkinsonian mesencephalic rodent model.

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Experiment Monkey Model

• Samiri sciureus, Squirrel monkeys weighing 0.7
  1.0 kg
• Baseline locomotor activity was measured using
  computerized movement monitor cage containing
  infrared sensors.
• 2 mg/kg treatment of saline or
• 1-methyl-4-phenyl-1,2,3,6-tetrahydropteridine
  (MPTP) subcutaneously.

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• 2 ½ weeks after treatment with MPTP, locomotor
  activity was again monitored
• The monkeys were killed and the effects of MPTP
  were measured.

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Tissue Samples

• Tissue samples were cut into blocks 6mm thick.
• Dopamine and Homovanillic acid (HVA) were
  extracted and measured from the caudate and
  putamen (from the basal ganglia) using 0.4 N
  perchloric acid and chromatography.

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Results The effects of MPTP

• A decline in motor activity decreased more than
  50 when compared to the saline control.
• The levels of dopamine and HVA were reduced by
  50 when compared to the saline.

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Distribution of subunit mRNA

• Objective
• Significance

• To investigate the distribution of ?4, ?6, ?7,
  ?2, ?3 and ?4 mRNA in the Squirrel monkey basal
  ganglia.
• To observe the subunits greatly affected during
  degeneration and nicotine treatment.

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• ?4 and ?2 are predominant subunits in neuronal
  nicotinic receptors
• ?7 primary subunit in bungarotoxin (BGT)
  nicotinic receptors
• ?6 and ?3 have a unique distribution in mice.

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Distribution of subunit mRNA

• The blocks were further cut into 20?m thick
  sections to carry out in situ hybridizations
• Thawed onto poly-L-lysine coated slides and
  stored at -80C.

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• cDNA of the different subunits found in large
  cytoplasmic loops were used to sequence cRNA
  probes.
• A transcription Kit was used to carry out
  transcription. ?35SUTP was used as a label.

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• Slides with hybridized cRNA were placed against
  Hyperfilm ?-max with 14C-radioactive standards.
• Development time 6-8 weeks
• The optical and photographic density of the
  labeled probes were determined using Computer
  densitometry.

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Results Subunit mRNA after MPTP

• The lesions formed were similar to those in found
  in the early stages of Parkinsons disease.
• There were no changes in ?4, ?7, ?2 and ?4 mRNA
  in the substantia nigra.
• ?6 mRNA increased and ?3 mRNA decreased

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Mesencephalic culture model

• Objective

• To compare the distribution of receptor subunits
  using immuno-stains.
• To observe the effects of MPP (a metabolite of
  MPTP) after nicotine treatment.

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Mesencephalic culture model

• Ventral mesencephalon portions of E15
  Sprague-Dawley mice.
• dissected and the tissue cultures were prepared
  like the those extracted from the monkeys.
• Cut into 6mm thick blocks
• Dopamine and HVA extracted

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• Samples were centrifuged and incubated in 0.5
  trypsin-ethylenediaminetetraacetic acid.
• Culture medium stop further reactions.
• Centrifuged re-suspended in 5ml of the culture
  media.

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• Then they were plated on poly-D-Lysine Nunclon
  48-well dishes at a density of
• 3 X 105 cells/cm2.
• Added 125I?-BGT (bungarotoxin) was to intact
  cultures to measure the activity of ?7 containing
  receptors.
• Added 3HEpibatidine to membranes prepared from
  the culture to measure the activity of ?2 and ?6
  containing receptors

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• Tyrosine hydroxylase immunochemistry was used to
  measure the number of dopaminergic neurons.
• Special culture media consisting of goat serum,
  bovine serum albumin, polyvinyl pyrrolidone and
  Triton-X-100, PBS.

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• Left for 1 hr at room temperature
• Tyrosine hydroxylase was added and left overnight
  at 4?C
• Washed and then bound to immunoglobulins

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• Immuno-stained cells were counted under 100x
  magnification.
• 24 hrs prior to exposure to MPP, samples were
  incubated in 10?M nicotine.
• 3 ?M of MPP (active metabolite of MPTP) was
  added to dopaminergic neurons and compared to the
  tissue samples before they were exposed to
  toxins.

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Results Monkey vs Mouse Distribution of subunit
mRNA

• The distribution of subunit mRNA in the Caudate
  and Putamen in the monkey model
• ?4 and ?7 nicotinic mRNAs were present
• Low density of ?2 subunits and high densities of
  ?4 subunits were observed .
• ?4, ?6, ?2 and ?3 mRNA was found in the rodent
  mesencephalic culture model.

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Results Monkey vs Mouse Distribution of subunit
mRNA

• ?4, ?6, ?7, ?2, ?3 and ?4 mRNA was found in the
  monkey substantia nigra.
• ?4, ?6, ?2 and ?3 mRNA was found in the rodent
  mesencephalic culture model.
• The distribution of subunits in the mRNA in
  rodents and monkeys are similar

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Receptor subunits
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Results Mesencephalic culture

• The effects of neurotoxicity MPP on
  mesencephalic culture was measured by comparing
  the number of neurons before and after the
  application.

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• Before MPP the number of cells immunostained.
• 3Hepibatidine 2.2 0.4fmol/106 cells
• 125I ?-BGT 0.5 0.1 fmol/106 cells
• There was a 80-95 destruction in the
  nigrostriatal in the rodent mesencephalon model.
• After pre-nicotine treatment there was a 20
  decrease in dopaminergic cells.

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Conclusion

• Nicotinic receptors are cholinergic receptors
  that are made up of subunits that affect the
  activity of the receptor.
• There is some similarity in the distribution of
  subunits in brain of rodents and monkeys,
  however, the levels of expression differs.
• Locomotor activity decreases with MPTP toxicity.

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• MPTP and MPP toxicity reduces the number of
  dopaminergic neurons.
• Pre-treatment of nicotine reduces the loss of
  dopaminergic neurons caused by MPP
• The neuroprotective effects may be due to the
  activation of receptors containing specific
  subunits.

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References

• Court, J.A., Ruiz-Martin, C., Graham, A., and
  Elaine Perry. (2000) Nicotinic receptors in human
  brain topography and pathology. Journal of
  Chemical Neuroanatomy Vol 20 281-298
• Mihailescu, Stefan Drucker-Colin, Rene (2000)
  Nicotine, brain nicotinic receptors and
  neuropsychiatric disorders. Archives of Medical
  Research Vol 31 131-144
• Bondy, S., Ali, S.F., and M. Kleinman (2000)
  Exposure of mice to tobacco smoke attenuates the
  toxic effect of methamphetamine on dopamine
  systems. Toxicology Letters Vol 118 43-46
• Costa, G., Abin-Carriquiry, J.A. and Federico
  Dajas. (2001) Nicotine prevents striatial
  dopamine loss produced by 6-hydroxydopamine
  lesion in the substantia nigra. Brain Research
  Interactive Vol 888 336-342.
• Encyclopedia Britannica. www.britanica.com

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• Quik, M. and G. Jeyarasasingam (2000) Nicotinic
  receptors and parkinsons disease. European
  Journal of Pharmacology Vol 393 223-230.
• http//www.emory.edu/CHEMISTRY/justice/chem190j/ni
  cotinic_receptors.htm
• http//www.nida.nih.gov/meetsum/nicotine/slides/14
  Kellar/KellarSlides.html