SBIR/STTR Award attributes
Abstract A general mechanism for neurodegeneration, including Parkinsonandapos;s disease and Alzheimerandapos;s dementia, is the breakdown and subsequent aggregation of misfolded neuronal proteins. In the “synucleinopathies” for example neurotoxicity is associated with the cleavage of mis-folded alpha-synuclein (ASYN), probably mainly by asparagine endopeptidase (AEP) [Zhang et al 2017]. This generates neurotoxic peptides that then aggregate with ASYN in lysosomes, forming the Lewy inclusion bodies associated with neurodegeneration. A very similar mechanism exists in plant cells in which plant AEP breaks down misfolded proteins to produce vacuolar aggregates associated with programmed cell death (PCD) [Hatsugai et al, 2015]. However, plants do not contain ASYN, so, in order to mimic ASYN toxicity, plant cells were transformed to express the misfolding- prone A53T variant of human ASYN (phase I). Thymoquinone was then used to trigger plant PCD [Hassanien et al 2013] and the plant cells expressing ASYN-A53T were shown to be significantly more susceptible to this toxicity than controls. In phase II a mutant population of these transgenic (ASYN) plant cells will be selected for survival under this procedure. This is an example of “target-directed evolution” in which mutants that survive selection should “evolve” toward increased biosynthesis of metabolites that inhibit AEP and/or ASYN toxicity. Individual mutant plant cell clones with ASYN-protective activity will be identified by screening extracts of resistant cultures, and micro-analytical methods [Kelley et al, 2019] will then be used to identify active metabolites as leads. Lobelia cardinalis cell cultures were used in phase I because we had previously transformed these with the human dopamine transporter gene to mimic MPP+-induced dopaminergic neurotoxicity [Brown et al 2016]. In phase II we will also use the medicinal plant, Polygonum multiflorum, which contains a stilbene that inhibits ASYN toxicity [Zhang et al 2018]. Phase II aims to identify novel leads that engage the AEP and ASYN targets, and to test the most promising of these in cellular and animal models of synucleinopathy. Leads will be developed with the University of Kentucky Parkinsonandapos;s Disease Research Center and a pharmaceutical partner. Identification of leads that engage these targets will also support target- directed evolution in mutant plant cells as a commercial platform for drug discovery.Project narrative Several human neurodegenerative diseases, including familial Parkinsonandapos;s disease, share a mechanism in which the enzymatic modification and subsequent aggregation of a nerve cell protein called alpha-synuclein kills nerve cells. Plant root cells contain a similar enzyme, but they do not contain alpha-synuclein. By creating plant cells expressing human alpha-synuclein the applicants will be able to seek novel plant metabolites that inhibit this neurotoxic process, and that will lead to novel drugs to treat these devastating neurodegenerative diseases.
