Effects of Pharmacological Agents upon a Transgenic Model of Parkinson

1
Effects of Pharmacological Agents upon a
Transgenic Model of Parkinsons Disease in
Drosophila melanogaster

• Pendleton, R., F. Parvez, M. Sayed, and R
  Hillman. 2002. Effects of pharmacological agents
  upon a transgenic model of Parkinsons Disease in
  Drosophila melanogaster. Journal of Pharmacology
  and Experimental Therapeutics 300(1) 91-96.
• Jeff Kufel Bio 475 (01)

2
Parkinsons Disease, What Is It?

• a chronic progressive nervous disease chiefly of
  later life that is linked to decreased dopamine
  production in the substantia nigra.
• Second most common Neurodegenerative Disease
  affecting over 1 million people in North America
  and 1 of the population over 50.

3
What is a Neurodegenerative Disease?

• Neurodegenerative diseases are those which are
  characterized by the progressive and irreversible
  loss of neurons from specific regions in the
  brain.

4
What is a Neurodegenerative Disease? (continued)

• In the case of Parkinsons Disease, losses in the
  portion of the brain known as the basal ganglia
  (more specifically the substantia nigra) result
  in abnormalities in the control of movement.

5
Basal Ganglia

• Portion of the brain that controls voluntary
  movement.
• Made up of distinguishable interconnected nuclei
• Globus Pallidus (GP)
• Subthalamic Nuclei (STN)
• Substantia Nigra (SNC and SNR)
• Striatum

6
Substantia nigra

• The Substantia nigra is a layer of pigmented gray
  matter situated in the brainstem containing the
  cell bodies of dopamine producing nerve cells.
• 2D and 3D images come from the UPRES "Cortex
  cérébral et Epilepsies"/SIM Laboratory (Rennes
  University/France) Illustrations come from the
  Atelier d'Infographie of the Medical Imaging
  Department of Pr Carsin in the Rennes University
  Hospital.

7
What Are The Characteristics of Parkinsons
Disease?

• Loss of dopaminergic neurons in the substantia
  nigra.
• Formation of intraneuronal inclusions (Lewy
  Bodies).
• Extrapyrimidal (involuntary) movement Disorders

8
What Are Dopaminergic Neurons?

• Dopaminergic neurons are the neural cells which
  produce dopamine.
• In Parkinsons Disease, the dopaminergic cells
  deteriorate and this leads to reduced production
  of dopamine.

9
Major Neuronal Pathway In Basal Ganglia

• The picture on the left shows normal neuronal
  pathway.
• The picture on the right shows the pathway of
  someone with Parkinsons Disease.

10
What is Dopamine?

• Dopamine is a neurotransmitter (chemical
  messenger) in the nervous system.
• It acts to coordinate voluntary muscular
  activities through excitatory and inhibitory
  control.

11
Dopamine Synthesis and Release in Substantia Nigra
12
How Dopamine is Synthesized

• Dopamine is synthesized in the pre-synaptic
  terminal
• Tyrosine is converted to L-DOPA using the enzyme
  Tyrosine Hydroxilase
• L-DOPA is converted to Dopamine by the enzyme
  Aromatic Amino Acid Decarboxylase.

13
How Dopamine is Released

• After synthesis, Dopamine is taken up by synaptic
  vesicles
• Dopamine is released from these synaptic vesicles
  into synaptic cleft.
• After release, the Dopamine binds to receptors on
  the post-synaptic terminal, propagating the
  action.
• The remaining molecules (not binding to
  receptors) are taken back into the synaptic
  terminal by transporters.

14
What are Lewy Bodies?

• Intracytoplasmic inclusions found in vacuoles of
  injured or fragmented neurons.
• The presence of Lewy Bodies indicates
  degenerative neuronal changes.
• A major component of Lewy Bodies is the
  pre-synaptic protein, Alpha-Synuclein.

15
Extrapyrimidal Movement Disorders Involve

• Bradykinesia
• Muscle Rigidity
• Resting Tremors
• Gait

16
Bradykinesia

• the slowing of voluntary movements initially,
  then generalized stiffness, fatigue, mild muscle
  pain progressing to generalized stiffness.

17
Muscle Rigidity

• Semi-flexed position of the hands, head, arm, and
  trunk difficulty in straightening.

18
Resting Tremors

• Involuntary shaking of the extremities

19
Gait

• short steps and shuffling of the feet in order
  to maintain balance.

20
What Is The Rationale of the Paper?

• In this paper, the authors are using the common
  fruit fly as a model to show how the major
  classes of drugs currently used as treatment
  affect the progression of the disease.

21
How Was the Human Alpha-Synuclein gene added to
Drosophila?

• Plasmids containing the human wild type alpha
  synuclein gene were injected into fly embryos.
• The wild-type (transgenic) progeny were obtained
  from prior studies of Feaney and Bender at
  Harvard Medical School.

22
Wild-Type Progeny

• The expression of the wild-type (transgenic)
  alpha-synuclein is associated with the
  degeneration and loss of dopamine containing
  neurons in the fly central nervous system.
• From previous studies, it had been found that
  these flies have a decreased ability to perform
  movement-related tasks.

23
How Were the Drosophila Tested?

• The Drosophila were tested using a negative
  geotaxis (climbing) assay over a specific time
  period
• The control used were Canton S flies
• Genetically normal, have constant breeding stock
  and the functional behavior is identical in the
  first few days of life.

24
What is the Climbing Response in Transgenic and
Control Flies?
25
How were the Tests Performed?

• Flies were added to flasks containing an agar of
  corn meal, corn syrup, water and dried yeast.
• These flasks were then emptied, leaving the pupa
  to emerge as adults.
• The next day (and 3-4 day intervals after), the
  adults were tested using the geotaxis assay.

26
Geotaxis (Climbing) Assay

• Flies were transferred into empty vessels.
• Each vessel had a line drawn 8 cm from the
  bottom.
• The flies were knocked to the bottom of the
  vessel and after 10 seconds, the number of flies
  that crossed the line was recorded.

27
Geotaxis (Climbing) Assay continued

• Both transgenic and Canton S flies were placed in
  drug-treated food for 13 days and the same test
  was performed.

28
What Drugs Were Used?

• L-Dopa
• Pergolide and Bromocriptine
• SKF 38393
• Atropine
• Alpha MT and p- chlorophenalanine (PCPA)

29
L-DOPA Results

• L-Dopa reversed the deteriorating motor
  activities of the transgenic flies.
• It did not affect climbing activity in the
  controls.
• The increased L-DOPA in the neuronal pre-synaptic
  cells allowed for greater conversion to dopamine
  by the enzyme Aromatic Amino Acid Decarboxylase.

30
L-DOPA continued

• Shaded portion Canton S
• White portion UAS-wild-type -alpha synuclein.
• Wild-type Control Group had 3.8 flies which
  escaped.
• Flies treated with L-DOPA had 7.7 flies escape.
• No effect on the control normal flies

31
Bromocriptine and Peroglide Results

• Dopamine receptor agonists.
• Act on the Dopamine inhibitory receptor pathway
  (D2).

32
Bromocriptine and Peroglide

• Shaded portion Canton S
• White Portion UAS-wild type
• alpha synucein
• Wild-type Control group had 3.9 flies escape
• Flies treated with Bromocriptine had 6.3 flies
  escape
• Flies treated with Peroglide had 6.9 flies escape
• No effects on the control normal flies

33
SKF 38393 Results

• Also a Dopamine receptor agonist.
• Acts on the Dopamine excitatory pathway (D1).

34
SKF 38393 continued

• Shaded Area Canton S
• White Area UAS- wild type-
• alpha synuclein
• Wild-type control had 4.0 flies escape
• Flies treated with SKF 38393 had 8.4 flies
  escape
• No effect on the control normal flies

35
Atropine Results

• Atropine is a muscarinic receptor antagonist.
• It is a drug that binds to receptors in the
  parasympathetic nervous system and effectively
  blocks them through competitive inhibition.
• The parasympathetic nervous system acts to
  maintain normal body function.

36
Atropine continued

• Shaded Area Canton S
• White Area UAS-wild-type alpha
• Synuclein
• Wild type control had 4.0 flies escape
• Flies treated with atropine had 5.7 flies escape
• No effect on control normal flies

37
Alpha MT and PCPA Results

• Alpha MT and PCPA (p-chlorophenalanine) both act
  as inhibitors of enzymes in dopamine synthesis.
• In the case of PCPA, it also inhibits the
  synthesis of serotonin (a major biogenic amine in
  Drosophila)

38
Alpha MT and PCPA continued

• Shaded Area Canton s
• White Area UAS-wild-type
• Alpha synuclein
• Wild type control had 3.9 flies escape
• Flies treated with PCPA had 2.4 flies escape
• Flies treated with Alpha MT had 1.1 flies escape.
• There was a significant change in the control
  indicating a decreased response in both groups
  compared to the untreated group.

39
Conclusion

• The aim of these tests was to see if the drugs
  currently used in treatment of Parkinsons
  Disease increase the negative geotaxis in
  Drosophila melanogaster.
• L-DOPA, Peroglide, Bromocriptine, SKF 38393, and
  Atropine all resulted in some success in the
  increase of negative geotaxis.

40
Conclusion continued

• Alpha MT and PCPA were used as negative controls
  and did not produce any beneficial effects.
• Since the Drosophila model mimics the impairments
  of Parkinsons Disease, this model will be
  beneficial in the development of new drugs in
  order to combat the disease.