Download Lewy Bodies in PD.

Lewy Bodies in PD.
Lewy Body Inclusions and the
effect of coffee and decaffeinated
extracts on Drosophila neuronal
cultures
Introduction to PD
• Parkinson’s disease is a
chronic neurodegenerative
disease characterized by a
variety of motor and nonmotor symptoms with no
proven successful therapy.
• Typical hallmarks of PD are:
1.
2.
Loss of Dopaminergic
neurons in the Substantia
nigra pars compacta
region of the brain leading
to motor disorders.
Presence of LB in the
surviving Dopaminergic
Neurons.
Two Types of Parkinson’s disease
• Sporadic PD: Non-inheritable,
onset due to chemical insults
resulting in mutations in
associated genes such as
LRRK2 or SNCA genes
• Familial PD: Mainly associates
to Autosomal dominant
mutations in genes such as
SNCA.
• A53T: increases propensity of
Fibrillar LB formations
• E46K: increases Propensity of
Protofibrils or Oligomeric asyn to form
• A30P: accelerates
protofibrillar formation.
What is Alpha-synuclein
• Alpha synuclein is the culprit of LB
formation
• It is:
1.
2.
3.
140 amino acid protein. The αsyn has a 12-amino acid stretch
in the hydrophobic core and is
concerned with the fibrillar
aggregation in DA cells.
Natively unfolded protein with a
hydrophobic region and an
acidic tail as is the same with
beta and gamma synuclein.
Believed to be extensively
localized in the nucleus and
found in the synapse thus the
name synuclein. But its function
is still under study.
Mark R. Cookson, The Biochemistry of Parkinson’s disease
What is LB from other science journals
• Lewy bodies are fibrillar
protein aggregations found in
surviving dopaminergic
neurons in PD.
• Its main constituents of
interest are α-syn and ubiquitin
and β amyloid fibrils.
• LB forms from Oligomeric α-syn
which arises from monomeric αsyn.
• Some papers say the oligomeric
form is toxic which disrupts
cellular permeability and LB is a
cellular rescue response; others,
LB aggregations trigger aptosis.
• Even though LB are associated
with fibrils which can be
stained with immunogold,
However, it has been tested
that A-53T mutant form is able
to form aggregates by it self
Drasophila Melanogaster
• Why Drasophila melanogaster:
1.
2.
3.
Cheap and easy to maintain
High reproduction rate
Entire genome has been
sequenced
4. Readily manipulate gene
expression in the embryo
creating gain or loss-offunction conditions almost at
will.
A53T line is used because the
mutation accelerates LB
aggregation
Antibodies and dyes
• Experiments have been done
to show that the 1:5000 of
primary antibodies give the
best distinction between LB
and background signals
6.000
LB/BG Intensity
5.000
4.000
3.000
5.051
3.887
3.519
1 in 5000
1 in 7500
1 in 10,000
2.000
1.000
0.000
• DAPI: stains the nucleus
(Blue) (1:2000)
• Anti-HRP: stains the Na+K+
channels on cells membrane
(Green) (1:100)
• Primary antibodies: selectively
stain for a-syn (1:5000)
• Secondary antibodies:
selectively binds to primary
antibodies and emits orange
light. (1:2000)
Objectives:
• Define Lewy Bodies
• Quantify the number of LB in A53T cell line over
1.2.3.6 and 9 day-in-vitro (DIV) and total
number of DA cells in the same culture.
• Determine if LB is a cause or a rescue response
of PD.
Methodology:
• Eggling of A53T fruit flies for 4½ to 5 hours.
• Cells are extracted from the eggs at the right stage.
• Cells are grown in special mediums for differentiation in
to neuronal cells for specific time internal.
• Caffeine, decaffeinated, Tobacco and Quest drugs are
added at 3DIV.
• Immunocytochemistry
• Analysis: take images of all LB like aggregations and DA
cells.
• The size of average LB size for each batch is obtained
• The ratio of DA cells/LB is obtained for each treatment
group.
Results for control at 1,2,3,6 and 9DIV
14.000
12.000
10.000
8.000
Size (μm)
• Most significant
figure in this chart
is the average size
of LB over time
remains stable
starting from
2DIV.
Control: Average LB and DA cell
Sizes
6.000
N=34
N=61
N=21
N=18
Ave LB Size
Ave DA Cell Size
4.000
2.000
0.000
2
3
6
Days In Vitro
9
Control total #LB/DA Ratio
5.000
4.500
3.974
4.000
3.548
3.500
Total #LB/DA ratio
• Provided that
the sizes of LB
over time is
relatively stable
from 2 to 9DIV,
the ratio of total
number of LB
cells versus
#DA is also
stable.
However, the
ratio for 1DIV is
3 times higher.
This
information
tells me that the
formation of LB
is between 1DIV
and 2DIV.
3.317
3.000
2.799
2.500
2.000
1.500
1.000
0.500
0.077
0.000
1
2
Days In Vitro
3
6
9
Coffee and Decaff extract added at
Chart Title
3DIV
14.000
N=769/24
12.000
Total # LB/DA Ratio
• The addition of
coffee and decaff
extracts at 3DIV
reduces the
number of LB in
both cases.
• However, the
ratio declines
either because of
an increase of
LB as the drugs
are used up over
time or a
decrease in total
number of DA
10.000
N=615/72
N=823/102
8.000
Control
N=276/50
6.000
Coffee
Decaff
4.000
2.000
0.000
6
9
Days in Vitro
Conclusion:
• Lewy Body is filamentous protein aggregation with a size
range of 4 to 7μm which mainly constitutes α-synuclein and
it is up regulated early in drosophila neuronal cultures.
• From these experiments, the formation of LB is studied
in both the absence and presence of neuroprotective
drugs.
• The number of LB increases drastically between 1DIV
and 2DIV and stays stable both in numbers and sizes.
• The data shows that both coffee and decaff extracts
reduce the number of LB after LBs are fully formed
which implies that these drugs are involved in the
degradation of LBs.
References:
•
•
•
•
Auluck et al 2010
Mel B. Feany and Welcome W. Bender. A drosophila model of Parkinson’s disease
Pavan K. Auluck, Gabriela Caraveo and Susan Lindquist. Α-synuclein: Membran Interactions and Toxicity In Parkinson’s
Disease.
Walter J. Schulz-Schaeffer. The synaptic Pathology of α-synuclein aggregation in dementia with Lewy bodies, Parkinson’s
disease and Parkinson’s disease dementia.