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Of neurotoxicity and α -synuclein

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Of neurotoxicity and α -synuclein. Richard Wilson. Clayton DF & George JM (1999) J Neurosci Res 58 , 120–129. Presentation outline. Motivation for the miniproject Neurons and disease The pore hypothesis The study Results Discussion. Motivation. No – not Catholic fervour…  - PowerPoint PPT Presentation

Text of Of neurotoxicity and α -synuclein

  • Of neurotoxicity and -synucleinRichardWilsonClayton DF & George JM (1999)J Neurosci Res58, 120129

  • Presentation outlineMotivation for the miniprojectNeurons and diseaseThe pore hypothesisThe studyResultsDiscussion

  • MotivationNo not Catholic fervour but a desire to combat Parkinsons disease (PD) Second most common neurological disease in the elderly (in the western world)PD symptomsProgressive loss of motor function: difficulty in initiating movements, rigidity, staggering, resting tremorNo cure available

  • Neurons and PDNeurons are specialised cells forming the nervous systemAll neurons produce neurotransmittersDopaminergic neurons produce dopamineDopamine is essential for motor controlPD results from destruction of dopaminergic neuronsBut what is killing these cells?

  • Lewy bodiesNeurons of PD patients contain characteristic inclusions called Lewy bodiesvisible under the light microscopehttp://medweb.bham.ac.uk/

  • The cause of PD?Lewy bodies (LBs) are largely composed of fibrillar aggregates of the protein -synuclein -Synuclein in its normal role (its native form) is unstructured and not aggregatedThis has prompted research into how its structure is related to toxicityInitially LBs thought to be toxicNow believed that LBs are at least benign if not a protective response to the diseaseProtofibrillar -synuclein: an intermediate form between native and fibrillar is the suspect

  • Pore hypothesisVolles & Lansbury (2003) proposed that pore-like protofibrils may puncture the cell membrane causing leakage of vital molecules and cell deathVolles MJ & Lansbury PT (2003)Biochemistry 42 (26), 7871-7878250 nm squareAtomic force microscopy imageGoldberg MA & Lansbury PT (2000)Nature Cell Biology 2, 115-119

  • This studyAim: to test the neurotoxicity of various forms of -synucleinMethodInduce protein aggregationCulture cellsAdd protein to cellsTest for toxicityThe experiments were performed in vitro (actually in plastic) usingartificially synthesised (recombinant) -synucleinB104 rat neuroblastoma cells (from central nervous system)

  • 1) Inducing protein aggregation-Synuclein incubated according to published conditions (Hoyer et al 2002)250 M protein incubated at 37 degC with shakingat pH 4: 20 M citric acid buffer, sample taken at 1, 3 and 6 hoursat pH 7: 10 M phosphate buffer, sample taken at 24, 72 and 96 hoursSeveral techniques were used to check structural changes including electron microscopyHoyer W, Antony T, Cherny D, Heim G, Jovin TM & Subramaniam V (2002) J Mol Biol 322, 383-393

  • Incubated protein structurepH 4 protein amorphous aggregatespH 7 protein protofibrillar aggregates

  • 2) Culturing cellsCells introduced to rows of 6 wells in 96-well plates4 plates of cells cultured in medium with antibiotics for 4 days at 37 degC in an incubator:2 plates with serum to give undifferentiated cells2 without serum to give differentiated cells (more like adult neurons)A 96-well plate

  • 3) Adding protein to cellsFor each plate, 3 control rowsmedium onlycells plus buffercells plus Tween 20% (kills cells)and 3 experimental rows cells plus 1 M proteincells plus 5 M proteincells plus 10 M proteinPlates incubated for 24 hours at 37 degC in 10% CO2 humidified atmosphere

  • 4) Toxicity testing: MTT assay3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the wellsPlates incubated for 4 hoursHealthy cells reduce pale yellow MTT to dark blue formazanLysis solution (15% SDS/50% N,N-dimethylformamide) added to wells to release the formazan from the cellsPlates 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 proteinsNo toxicity(!)

  • Results pH 7 proteinsEnhanced function!Toxicity?

  • DiscussionThe results are unexpected sinceprevious studies* found both native and fibrillised -synuclein to be neurotoxicAnd surprising asthe function of undifferentiated cells was enhanced by -synuclein protofibrilsEnhancement has not been observed beforeHowever, 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, 2744127448

  • Implications for PD theoryIf these tentative results were confirmed, then it is clear thatprotofibrils dont puncture the cell membraneBut, of course, protofibrils may attack vesicles or mitochondrial membranesOne other possibility is that B104 cells are not a good model for PD

  • AcknowledgmentsBiological SciencesTeresa Pinheiro, supervisorBruno Correia, mentorNarinder Sanghera, cell wizardEPSRC, essential fundingMOAC: thanks for your support

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