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Nicotinic receptors and parkinson’s disease By: Maryka Quik and Jeyarasasingam European Journal of Pharmacology 393 (2

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Nicotinic receptors and parkinson’s disease By: Maryka Quik and Jeyarasasingam European Journal of Pharmacology 393 (2000): 223-230 Presented by: Natasha Frett. Introduction. Parkinson’s disease is a neurodegenerative disease that results from loss of dopaminergic neurons.

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slide1
Nicotinic receptors and parkinson’s disease

By: Maryka Quik and Jeyarasasingam

European Journal of Pharmacology 393 (2000): 223-230

Presented by: Natasha Frett

introduction
Introduction
  • Parkinson’s 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.
slide3
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

slide4
Acetylcholine at dentritic portion of neuron is received by nAChR
  • allows the influx of Ca 2+
  • Causes release of synaptic vessicles
slide5
Nicotine
  • agonist
  • decreases receptor breakdown
  • inhibits monoamine oxidase B, the enzyme that breaks down dopamine
slide6
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
slide7
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
protective subunits
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
slide9
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.
experiment monkey model
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.

slide11
2 ½ weeks after treatment with MPTP, locomotor activity was again monitored
  • The monkeys were killed and the effects of MPTP were measured.
tissue samples
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.
results the effects of mptp
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.
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.

Distribution of subunit mRNA
slide15
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.
distribution of subunit mrna1
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 -80°C.
slide17
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. [35S]UTP was used as a label.
slide18
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.
results subunit mrna after mptp
Results: Subunit mRNA after MPTP
  • The lesions formed were similar to those in found in the early stages of Parkinson’s disease.
  • There were no changes in 4, 7, 2 and 4 mRNA in the substantia nigra.
  • 6 mRNA increased and 3 mRNA decreased
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.

Mesencephalic culture model
mesencephalic culture model1
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
slide22
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.
slide23
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 [3H]Epibatidine to membranes prepared from the culture to measure the activity of 2 and 6 containing receptors
slide24
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.
slide25
Left for 1 hr at room temperature
  • Tyrosine hydroxylase was added and left overnight at 4C
  • Washed and then bound to immunoglobulins
slide26
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.
results monkey vs mouse distribution of subunit mrna
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.
results monkey vs mouse distribution of subunit mrna1
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
results mesencephalic culture
Results: Mesencephalic culture
  • The effects of neurotoxicity MPP+ on mesencephalic culture was measured by comparing the number of neurons before and after the application.
slide30
Before MPP+ the number of cells immunostained.
    • [3H]epibatidine 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.
conclusion
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.
slide32
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.
references
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
  • Quik, M. and G. Jeyarasasingam (2000) Nicotinic receptors and parkinson’s disease. European Journal of Pharmacology Vol 393: 223-230.
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