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

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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

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3 hours

6 hours

Results – pH 7 proteins

Enhanced function!

Toxicity?

pH7 proteins - undifferentiated cells

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0 µM 1 µM 5 µM 10 µM

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Native

24 hours

72 hours

96 hours

pH7 proteins - differentiated cells

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Native

24 hours

72 hours

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