 Well thank you very much, Julio, for the invitation to come and speak here and also for being so generous in including Parkinson's disease and translational psychiatry. I think that's very generous. So the role of alpha-synuclein became of interest when the gene for alpha-synuclein, which is SNCA, was found to have mutations which caused a rare heritable form of autosomal Parkinson's or PARC1. And shortly after that it was discovered that alpha-synuclein was one of the core proteins in the Lewy body which is commonly seen as the defining pathological marker of, histopathological marker of Parkinson's disease. But some time after that there was the discovery that in fact duplications or triplications within the SNCA gene itself, leading to increased expression of alpha-synuclein, caused more severe disease. And it led Singleton, who was one of the discoverers of this observation, to make the hypothesis or observation I suppose that if 100% increase in SNCA expression causes disease onset in the 30s and a 50% increase in expression leads to disease onset in the 40s, smaller increases in alpha-synuclein expression might be central to typical idiopathic Parkinson's disease. So there's been quite a lot of interest in trying to find evidence that this is true. And it's been difficult to do this because opportunities to look at increased expression in the brain only come about at post-mortem when indeed often there is an exhaustion of the nigral neurons and so it is not clear what the relationship is going to be between SNCA triplications and gene expression. GWAS has been of interest in the two, but also some disappointment because it's really only identified these locations here with the REP1 and also changes in the 3-prime UTR region leading to changes in stability of SNCA. And both of these could conceivably alter expression of the gene and multiplications that I've spoken about and also downstream signals such as LAC2, TAU and possibly FMR1. So the idea that increased expression might occur in sporadic disease is still there but hasn't actually been nailed down. Now I've just put this slide in to state the obvious that it's not necessarily all about translation which can produce dose and product, but there's increasingly the evidence that other park genes and those that have been also related to increased risk of disease are going to alter both the function and the secretion and the clearance of alpha-synuclein and may also have effect on endosome and exosome trafficking as well as the auto-phagian ubiquitinization of alpha-synuclein. So there's many sites at which expression could be altered other than simply at translation. So the idea that we could actually measure alpha-synuclein as an extracellular protein came about really because it was observed in the condition medium of cell cultures and this is surprising perhaps because it had been there prior to that scene as entirely as a cytosolic protein because it doesn't have a leader sequence and normally exists as a natively unfolded protein. But it had been found in CSF and a series of reports came out roughly at the same time and this is a result that came from a collaboration between Jiaxin Li and Collins Group and ourselves and Jiaxin showed that if one looks at just the monomer in plasma that it's reduced in Parkinson's disease and at the same time other groups had shown that monomer was reduced in CSF and there had been varying results as to what was the finding in plasma. Some had said it was high and some had said it was low. And this had seemed a contentious issue and one that was not clear as to how to resolve it. So we addressed this by attempting to develop an ELISA that might give a more sensitive and higher three-put analysis and we developed our own antibody for the capture and fiddled around with the mechanisms and particularly the use of detergents which coming back to Collins' observation about the importance of alpha synuclein in lipid, particularly the lipids of cells in the blood, seemed to be important. And so when we looked at the same cohort of subjects, age-match controls in Parkinson's disease and did the monomer, we found a very similar result as prior. But when we used the same subjects to do the ELISA, we found that, in fact, the level was high, suggesting that we were seeing different species of alpha synuclein using the ELISA and the monomer. And I'm not going to spend a lot of time as to what that is, but I'm happy to discuss it if people would like. But probably we're seeing a tetramer of alpha synuclein or something at about 56 kilodaltons. So we then looked at a much larger cohort using healthy controls from the ABL group with 720 subjects and 150 subjects who had been with Parkinson's disease who had been fully phenotyped and effectively confirmed the findings that, in fact, on average, statistically there was a higher level, but you can see the separation is not clean and complete. If we plot this in a different way in which the level of alpha synuclein is along the axis here and the percentile is plotted in this direction and this is the healthy controls here. And the dotted line, the red dotted line is the Parkinsonian population. And you can see that roughly, if we look at it this way, that about a third of the Parkinsonian population sit above the 90th percentile of healthy controls. And we use this to use a risk ratio estimate of the risk of getting Parkinson's disease by saying, well, with a population such as this and using the known incidence of Parkinson's disease in the population, what's the odds of having this ratio of Parkinsonism for this particular level of alpha synuclein? And these large blue dots, we could produce a risk curve which shows that at the 90th percentile there's about a 39% increase in the risk of or a 0.39 odds ratio of getting Parkinson's disease. And if you curve fit this, the dotted line is the curve fit going on further up to show that the risk progressively increases at a higher level of alpha synuclein for getting Parkinson's disease. The second point that I think is of interest is that this gray line here that's sitting underneath the red one is the healthy control or the age match control population multiplied by 1.5. So what it's showing is that this is, that the Parkinson's disease curve is almost a complete match of roughly a 145% increase in alpha synuclein levels, suggesting that all of these are produced by a 145% increase in alpha synuclein levels all the way up here to produce this Parkinson's curve. And I'm going to come back to this question of the rise in alpha synuclein as a risk itself. And this, one of the ways in which we did this was to break the age match control group up into deciles of 80-year-olds, 70-year-olds, 60-year-olds, and a 40-year-old group, which I have to point out that the 40s and the 80s are the two weakest groups, is really because of the size of the populations. And I've, for, so that the curves are more obvious, I've chopped off the bottom and the, the top 95 to 100 percentiles and the bottom one so that you can see the spread. And this is the Parkinson's curve. And if we plot it a different way against the likely incidence of alpha, of Parkinson's in the population versus the percentage increase, you can see that there's a steady, linear increase in the incidence of, that there's a, sorry, a direct correlation between the incidence of Parkinson's disease and the percentage increase. In other words, the rise in alpha synuclein that comes with age predicts the rising incidence of Parkinson's disease in that age group. And so I think that this can be thought of in another way. Here is this percentage increase, which I showed you in this curve here. Plotted against age and you can see that this is rising in a steady manner as age occurs. Parkinson's is up here and it suggests, therefore, that something must happen in the rate of increase to actually cause Parkinson's to occur or to at least predict the risk in Parkinson's. So therefore it's the rate of rise, which I think is reminiscent a little bit about what Colin was talking about with A beta, that suggests that that's the transformation into Parkinson's disease. And whether you could plot Parkinson's right out here and suggest that if we lived to 130, you would all, we would all get it. Or whether, in fact, there's some rapid rise in the curve that occurs here is unclear. So the reason I think that we see, by a high level, causes the risk of Parkinson's therefore is because 145% increase of someone who's got a 200 nanograms of alpha-synuclein is going to put them right up here and therefore enrich this population for finding them. But probably if we're wanting to actually have an early detection, what we need to do is be serially measuring alpha-synuclein and look for someone who's got a sudden rise in the level. And therefore I think it's probably the rate of change that is more important than the absolute level itself, although one could spend a lot of time looking here to look for enriched populations and look for pre-symptomatic disease. The other point which helped to, I think at least in my mind, suggest that we've got something that is disease specific rather than a non-specific thing is when we looked at the mild cognitive impairment, the AD group from the ABLE study, we found that they also had a lower level of alpha-synuclein and so this wasn't a non-specific effect associated simply with neurodegeneration and gave us some heart that this was a real observation. The third factor which I think also points to the fact that we're looking at something that is perhaps got a biological basis is that when we sorted our alpha-synuclein group into those with high alpha-synuclein levels and this is defined as being greater than the 90th percentile of controls and those with low which is the people with less than the 75th percentile of controls and you might argue whether that's really low but we found that so the populations are the same age group, same disease duration but the people who had high levels, had much higher levels of anti-Parkinson's medications and they tended though not clearly statistically different to actually commence treatment earlier and they had higher levels of some of the markers of bradykinesia which were trending towards significance and perhaps with the higher population we might have seen some of this fall out but the tapping and the 14 meter walk are both markers of bradykinesia. Perhaps the other interesting thing is that we also found that there was a tend towards worse cognitive function with the higher levels and this is particularly measuring particularly if you pick out one part of the adenbrooks which is the word finding and the scope of COGAS well which is a broad measure of cognitive function in Parkinson's disease. Now I want to just step away from the previous question of whether alpha-synuclein therefore is perhaps a predictor of risk of Parkinson's disease and then talk a little bit about well where does it come from and I guess the inclination is to think well Parkinson's is a brain disease therefore it must be coming from the brain but even if and it seems likely that alpha-synuclein is high in the CSF of people with Parkinson's disease or tends to be higher if one thinks of the relative volume of CSF versus the relative volume of plasma there would have to be a very large transition of alpha-synuclein into the plasma or a very low rate of clearance for it to actually be high and alpha-synuclein is expressed in by blood cells, red blood cells, platelets and PBMCs and so one of the things we did first was to look at a group of subjects who are about to undergo bone marrow transplant so that they would be a period of time in which they had effectively a lymphopenia and no platelets and this is this subject group of subjects before the bone marrow ablation and this is after the bone marrow ablation and there's a roughly a 50% drop in alpha-synuclein. Our prediction is that the remaining was produced mostly by red blood cells which were unaffected. The second thing we looked at was to take a proportion of in a small number of healthy controls in Parkinson's to take B cells and transform them and at this stage it wasn't because we particularly thought the lymphocytes would be the guilty party but it was just that we could do it to B cells and we knew the technology and looked at the rate of apoptosis and found that that was higher in the B cells removed from the Parkinsonian subjects and I'm going to go back to that question again shortly and down here there's the just to show that this is in the PBMC which is in a small amount because of the effective album and other serum and plasma proteins but it's mainly in the PBMC fraction not in the granular sites but also in the red blood cells and the last thing that I want to do is to just spend a little bit of time discussing other evidence from the literature and this is a series of quite a lot of papers that I'm going to go through very quickly and the first is a paper by an Italian group which showed that this is alpha-synuclein levels in the white is the control groups and the black is in Parkinsonian subjects and you can see there's almost a 50% increase in B cells and T cells and natural killer cells this is a relatively small population of proportion of PBMC's and so it points to these cells as being the the likely source of increase in alpha-synuclein in the plasma second is that if one looks at alpha-synuclein expression levels and the this is in PBMC's and the percentage of cells expressing apoptotic markers in response to a steroid stress test that those with the highest death rates were also those with the highest alpha-synuclein expression levels this is a finding from Kim et al. The other factor is that people with Parkinson's disease have increased have reduced numbers of cells particularly T cells and B cells of these particular subgroup markers and this is both in terms of absolute numbers as well as percent percentage of blood cell counts and a particular target with the naive helper cells the CD4 CD25 CD45 RA positive group and these had increased phas ligand expression and this was only apparent in vivo and not in vitro which is makes it quite a difficult thing to study because we had originally gone about trying to study this in vivo in vitro and found it hard and the important point is that this is not a response to circulating dopamine because in fact dopamine does kill B cells. The other feature that is important is that lymphocytes from Parkinsonian patients show a number of features which we would normally think of being associated with the the midbrain of people with Parkinson's disease that is with mitochondrial complex deficits dysfunction of the ubiquitin proteasome system DNA damage increased activated caspase 3 and 9 and interestingly these oxidative stress is more likely to increase apoptosis in lymphocytes from PD patients and over expression of alpha-synuclein in lymphocytes also produces oxidative stress and apoptosis and finally the lymphocytes from subjects with parkin mutation were also highly sensitive to apoptosis again suggesting that there is a mechanism that's operating to kill lymphocytes in Parkinson's disease and that this is perhaps the origins of the elevated alpha-synuclein that we're looking at. Now I think there's several if this is true there's several things that I would draw from this the first is that it suggests that the mechanism that we're seeing here of elevated alpha-synuclein may be actually coming from a process which is affecting blood cells but if this accurately predicts what's happening in the brain it then perhaps raises the question that Parkinson's is a more ubiquitous disorder than just nigral neurons or brain neurons because it affects more than just nigral neurons in the brain and perhaps that if you have an increased rate of death in a cell group that can be rapidly replaced it really has a not any great significance to have increased turnover of blood cells or other cell groups but neurons do not cycle and so an increased susceptibility to apoptosis is a much more serious thing then. The second reason that I think it's important to actually understand this is that it actually means that if this is true that the lymphocytes then become an excellent model for actually understanding why does why and how alpha-synuclein is related to a pathology in a cell and to use this to model the events that might be occurring in neurons and I suppose the last thing which may be of also relevance is that targeting alpha-synuclein expression in the plasma or its manufacture by the blood or the survival of blood cells may be a useful high throughput screen for Parkinson's disease. So I'd like to just acknowledge a number of people who've been involved in this. Rogan Tinsley did a lot of the lab work and particularly was significant in developing the ELISA and the antibodies were produced by Jerry Shaw at the University of Florida. Kate Cotschert is a neurology colleague of mine who and she and I have but she in particular has carried most of the burden of phenotyping Parkinsonian subjects and Miles Prince provided the hematological studies and particularly the patients having bone marrow transplants and of course Aval produced the healthy controls and of course money came from all the usual sources. So thanks very much.