Mimicking synuclein toxicity in plant cells to identify novel neuroprotective leads

Abstract Alpha synucleinASYNis believed to play an important role in the pathology of several neurodegenerative conditionsincluding some forms of Parkinsonandapos s diseaseDementia with Lewy Bodiesand Multiple Systems AtrophyThe most recent hypothesis is that the ASYN proteinsparticularly genetic variants such as AT that are prone to misfoldingare cleaved by the lysosomal enzymeasparagine endopeptidaseAEPto generate peptides that promote ASYN aggregation and initiate programmed cell deathPCDin vulnerable neuronsZhang et alInhibition of AEPand of ASYN aggregationare therefore prime molecular targets for pharmacotherapy of thesesynucleinopathiessee Brundin et aland maybe other neurodegenerative diseasesChemical synthesis has yielded few leadsand this proposal will use a novel plant biotech platform to generate active compoundsIn this approachplant root cells are transformed by expression of human neuronal proteins to make them susceptible to a specific mechanism of neuronal toxicityMutants of these transgenic plant cells are then selected for survival when exposed to this neurotoxic mechanismThis mutagenesis and survival selectionevolvesplant secondary metabolism toward biosynthesis of metabolites that inhibit the neurotoxic mechanismProof of concept used expression of the human dopamine transporterhDATin plant cellsThis makes the plant cells highly susceptible to cytotoxicity induced by the dopaminergic neurotoxinMPPwhich is accumulated by the hDATWhen transgenichDATmutants are selected for survival in MPPthe resulting sub population includes many individuals that overproduce known or novel metabolites that inhibit the hDATand or the cytotoxic mechanisms of MPPBrown et alAs regards the AEP ASYN mechanism of neurotoxicityplant cells naturally contain a homolog to lysosomal AEPwhichlike the human neuronal AEPis linked to PCDHatsugai et alMetabolites that regulate this process almost certainly exist in plantsand so mutant plant cells that survive AEP activation should include many that overproduce natural inhibitors of plantp AEPBecause plant and humanh AEP are homologsthese metabolites are potential neuroprotective leadsHoweverplant cells do not naturally contain homologs of humanh ASYN soto mimic the proposed mechanism of synucleinopathies we will create transgenic plant cells that express the hASYN variant ATBecause pAEP has similar substrate specificity to hAEPand because the peptides produced by hASYN AT cleavage are highly cytotoxicthese transgenichASYNATplant cells should show increased susceptibility to PCD when pAEP is activatedto be established in Phase IConsequently transgenichASYN ATmutants that survive AEP activation should include many individual clones in which inhibitors of pAEPor of peptide induced hASYN aggregationare over producedIn addition tonaturalmetabolitesmutation and selection shouldevolveplant secondary metabolism toward novel metabolites with greater inhibitory activityThis will be established in Phase II using conventional in vitro screensidentifying active metabolites by assay guided fractionationThe objective is to develop these active plant metabolites as therapeutic agentsor leads for synthetic modificationby the applicants in partnership with a major pharmaceutical companyThe application of the proprietary biotechnology to these important targets will also strengthen its commercial importance as a plant drug discovery platform Project narrative Several human neurodegenerative diseasesincluding familial Parkinson s diseaseshare a mechanism in which the enzymatic modification and subsequent aggregation of a nerve cell protein called alpha synuclein kills nerve cellsPlant root cells contain a similar enzymebut they do not contain alpha synucleinBy 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