Characterization of the amyloid precursor α-synuclein by ...
Of neurotoxicity and α -synuclein
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Transcript of Of neurotoxicity and α -synuclein
Of neurotoxicity and α-synuclein
RichardWilson
Clayton DF & George JM (1999)
J Neurosci Res58, 120–129
Presentation outline
Motivation for the miniproject Neurons and disease The pore hypothesis The study Results Discussion
Motivation
No – not Catholic fervour… but a desire to combat Parkinson’s disease (PD)
• Second most common neurological disease in the elderly (in the western world)
PD symptoms• Progressive loss of motor function: difficulty in initiating
movements, rigidity, staggering, resting tremor
No cure available
Neurons and PD
Neurons are specialised cells forming the nervous system
All neurons produce neurotransmitters Dopaminergic neurons produce dopamine Dopamine is essential for motor control PD results from destruction of dopaminergic
neurons But what is killing these cells?
Lewy bodies
Neurons of PD patients contain characteristic inclusions called Lewy bodies
visible under the light microscope
http://medweb.bham.ac.uk/
The cause of PD?
Lewy bodies (LBs) are largely composed of fibrillar aggregates of the protein α-synuclein
α-Synuclein in its normal role (its native form) is unstructured and not aggregated
This has prompted research into how its structure is related to toxicity• Initially LBs thought to be toxic
• Now believed that LBs are at least benign if not a protective response to the disease
• Protofibrillar α-synuclein: an intermediate form between native and fibrillar is the suspect
Pore hypothesis Volles & Lansbury (2003) proposed that pore-like
protofibrils may puncture the cell membrane causing leakage of vital molecules and cell death
Volles MJ & Lansbury PT (2003)Biochemistry 42 (26), 7871-7878
250 nm square
Atomic force microscopy image
Goldberg MA & Lansbury PT (2000)Nature Cell Biology 2, 115-119
This study Aim: to test the neurotoxicity of various forms of α-
synuclein Method
1) Induce protein aggregation2) Culture cells3) Add protein to cells4) Test for toxicity
The experiments were performed in vitro (actually in plastic) using• artificially synthesised (recombinant) α-synuclein• B104 rat neuroblastoma cells (from central nervous
system)
1) Inducing protein aggregation α-Synuclein incubated according to
published conditions (Hoyer et al 2002) 250 μM protein incubated at 37 degC with
shaking• at pH 4: 20 μM citric acid buffer, sample taken at
1, 3 and 6 hours• at pH 7: 10 μM phosphate buffer, sample taken at
24, 72 and 96 hours Several techniques were used to check
structural changes including electron microscopy
Hoyer W, Antony T, Cherny D, Heim G, Jovin TM & Subramaniam V (2002) J Mol Biol 322, 383-393
Incubated protein structure
pH 4 protein – amorphous aggregates
pH 7 protein – protofibrillar aggregates
2) Culturing cells
Cells introduced to rows of 6 wells in 96-well plates
4 plates of cells cultured in medium with antibiotics for 4 days at 37 degC in an incubator:• 2 plates with serum to give
undifferentiated cells
• 2 without serum to give differentiated cells (more like adult neurons)
A 96-well plate
3) Adding protein to cells For each plate, 3 control rows
• medium only• cells plus buffer• cells plus Tween 20% (kills cells)
and 3 experimental rows • cells plus 1 μM protein• cells plus 5 μM protein• cells plus 10 μM protein
Plates incubated for 24 hours at 37 degC in 10% CO2 humidified atmosphere
4) Toxicity testing: MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was added to the wells Plates incubated for 4 hours
• Healthy cells reduce pale yellow MTT to dark blue formazan
Lysis solution (15% SDS/50% N,N-dimethylformamide) added to wells to release the formazan from the cells
Plates read by automatic plate reader (absorbance measured at 570 nm)
Resulting data averaged and normalised to the positive control (cells plus buffer)
Results plotted as bar charts with standard deviation error bars
Results – pH 4 proteins
No toxicity(!)
pH4 proteins - undifferentiated cells
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0 µM 1 µM 5 µM 10 µM
MT
T r
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Native
1 hour
3 hours
6 hours
pH4 proteins - differentiated cells
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0 µM 1 µM 5 µM 10 µM
MT
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3 hours
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Results – pH 7 proteins
Enhanced function!
Toxicity?
pH7 proteins - undifferentiated cells
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0 µM 1 µM 5 µM 10 µM
MT
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pH7 proteins - differentiated cells
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0 µM 1 µM 5 µM 10 µM
MT
T r
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Native
24 hours
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96 hours
Discussion
The results are unexpected since• previous studies* found both native and fibrillised α-
synuclein to be neurotoxic
And surprising as• the function of undifferentiated cells was enhanced by
α-synuclein protofibrils
Enhancement has not been observed before However, the results are tentative because of
the lack of replication
*El-Agnaf et al (1998) FEBS Letters 440, 71-75; Sung et al (2001) J Biol Chem 276, 27441–27448
Implications for PD theory If these tentative results
were confirmed, then it is clear that• protofibrils don’t puncture
the cell membrane But, of course, protofibrils
may attack vesicles or mitochondrial membranes
One other possibility is that B104 cells are not a good model for PD…
Acknowledgments
Biological Sciences• Teresa Pinheiro, supervisor
• Bruno Correia, mentor
• Narinder Sanghera, cell wizard
EPSRC, essential funding MOAC: thanks for your support