1. Of neurotoxicity and α-synuclein Richard Wilson Clayton DF & George JM (1999) J Neurosci Res 58, 120–129

2. Presentation outline Motivation for the miniproject Neurons and disease The pore hypothesis The study Results Discussion

4. 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

5. 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?

6. Lewy bodies Neurons of PD patients contain characteristic inclusions called Lewy bodies visible under the light microscope http://medweb.bham.ac.uk/

7. 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

8. 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

9. This study Aim: to test the neurotoxicity of various forms of α- synuclein Method 1)Induce protein aggregation 2)Culture cells 3)Add protein to cells 4)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)

10. 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

11. Incubated protein structure pH 4 protein – amorphous aggregates pH 7 protein – protofibrillar aggregates

12. 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

13. 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% CO 2 humidified atmosphere

14. 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

15. Results – pH 4 proteins No toxicity(!)

16. Results – pH 7 proteins Enhanced function!Toxicity?

17. 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

18. 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…

19. Acknowledgments Biological Sciences Teresa Pinheiro, supervisor Bruno Correia, mentor Narinder Sanghera, cell wizard EPSRC, essential funding MOAC: thanks for your support