Potential Neuroprotection for Equine Pituitary Pars Intermedia Dysfunction

Abstract

Cushing's disease, known as pituitary pars intermedia dysfunction (PPID), is the most commonly diagnosed equine endocrinopathy. Compelling evidence suggests that alpha-synuclein, an abundant neuronal protein, can acquire a neurotoxic feature that kills dopamine secreting nerve cells in horse afflicted with PPID. Horseswith PPID have higher levels ofalpha-synuclein in the dopaminergic nerve terminals withinthe pars intermedia of the pituitary gland compared withhealthy horses. Using protein extracts from pituitary glands of horses with PPID, biophysical assays, and bioinformatic analysis, we have identified that equine alpha-synuclein has high propensity to convert (misfold and aggregate) into this toxic form. Our drug discovery lab focusses on identifying and designing small molecules to impede the formation of intermediate species and fibrils. Over the last decades, natural products have emerged as neuroprotective agents for chronic neurodegenerative diseases. We hypothesize that a natural product such as resveratrol locks equine alpha-synuclein molecules into a conformation unable to form cytotoxic oligomers and fibrils, thereby conferring cytoprotection. Most recently, we have identified the region 62-86 in the equine alpha-synuclein that is highly prone to convert into a toxic conformation. Fragment 62-86 is part of the non-amyloid-β component (NAC), a hydrophobic region that may contribute to alpha-synuclein oligomerization. The carboxy-terminal region (90-140) exhibits the lowest aggregation score. This region is intrinsically disordered and may contribute minimally to the aggregation. We conducted a pilot screening with silibinin and resveratrol (both flavonoids and natural products) on the different equine and human alpha-synuclein fragment peptides with thioflavin T fluorescence (ThT) assay. Resveratrol decreased relative ThT fluorescence of equine alpha-synuclein fragment 62-86 by ~70%. Treating equine alpha-synuclein fragment 62-86 with resveratrol reduced the number of alpha-synuclein fibrils as assessed by transmission electron microscopy. This project will set the stage to move into a lead optimization program and underscore the value of equine alpha-synuclein in PPID drug discovery.