Some future directions about α-synculein

The mechanism of the neurotoxicity of α-synculein is unknown; even the normal function of the protein is, at best, not clearly understood. An essential problem of current PD research is to remedy this deficiency. How can we ascertain the actual pathway of neurotoxity in synuclein-induced PD？

Forward/ chemical genetics: Perhaps most general in scope is forward genetical/chemical screening for reversion/enhancement of a neurotoxic phenotype in a dopaminergic transgenic model of PD. Such a method could identify pathway directly in line with synuclein, and so parallel pathways whose dysfunction affects neuronal survival against synuclein insult; it may be initially difficult to tell the difference. However, with a large number of interacting genes, one can begin piecing the abnormal physiology of synuclein together. I feel this approach is promising, partly because it makes very few tacit assumptions about the mechanism, other than that the transgenic model itself is valid. It also does not require any predetermined biochemical notions of the mechanisms. A lack of robust cell culture models (a situation which seems to be improving) in part explains why this approach has not been fully utilized to date.

Pure biochemistry: Although profitable thus far, I feel that a purely biochemical approach cannot take one very much further without knowing more about the biology of synuclein, that is, without know more about what to look at. This thesis has demonstrated that protofibrils can permeabilize acidic membranes, but the next essential question is whether this occurs in vivo, causing neural toxicity, or whether some other aggregation form, or the monomer, is the toxic entity. One can approach this difficult problem with biochemistry and cell biology, for example, via the introduction exogenous protofibrils into a cell culture model of PD, Or by searching for evidence of protofibrils in diseased tissue. My own preference would be to also pursue the problem reverse genetically using sequence variants of synuclein in a cell culture model.

Reverse genetics: This approach uses an α-synculein mutagenesis library such as the one described before to search for mutants which rescue or enhance a neurotoxic phenotype in PD model. What could we learn from such mutants? The toxicity of synuclein is probably due to a gain-of -toxic-function, making this special difficult question. One method for answering this question involves an in vitro study of the new mutant vs. wt, such as is described in this thesis. The necessary condition for this approach to be successful is that α-synculein toxicity exhibits an in vitro analog which we will observe. The “ will observe” is key: how to we know what properties to examine in vitro (this is the same problem which makes purely biochemical attack on PD difficult)? If aggregation is involved in disease, and there is evidence it is, it may be possible to observe such an analog by studying synuclein aggregation behavior alone, in solution. Additional candidate activities which could be checked include membrane binding/permeabilization, and other reasonable activities reported in the literature (phospholipase D inhibition for example). The forward genetic procedure will suggest further interactions/activities worth testing. From the opposite direction, Charter 4 of this thesis described the in vitro biochemical preselection of variants for reverse genetics. Placing the interesting variants (e.g., the slow fibrillizers) in a transgenic model would be potentially very revealing, which was an in vivo phenotype differing from that caused by the wild type protein observed. A negative result is also informative, by suggesting which in vitro properties are irrelevant to PD pathogenesis.