Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disease of aged horses that shows a similar pathophysiology as Parkinson’s Disease (PD) with increased levels of α-synuclein (α-syn). While α-syn is thought to play a pathogenic role in horses with PPID, it is unclear if α-syn is also misfolded in the pars intermedia and could similarly promote self-aggregation and propagation. Consequently, α-syn was isolated from the pars intermedia from groups of healthy young and aged horses, and aged PPID-afflicted horses. Seeding experiments confirmed the prion-like properties of α-syn isolated from PPID-afflicted horses. Next, detection of α-syn fibrils in pars intermedia via transmission electron microscopy (TEM) was exclusive to PPID-afflicted horses. A bank of fragment peptides was designed to further characterize equine α-syn misfolding. Region 62–87 of equine and human α-syn peptides was found to be most prone to aggregation according to Tango bioinformatic program and kinetics of aggregation via a thioflavin T fluorescence assay. In both species, fragment peptide 62–87 is capable of generating mature fibrils as demonstrated by TEM. The combined animal, bioinformatic, and biophysical studies provide evidence that equine α-syn is misfolded in PPID horses.
Transcriptional initiation is an important step in the regulation of transcription. When transcriptional initiation is dysfunctional, it will lead to abnormal gene expression. Overexpression of Serum Amyloid A (SAA) and self‐assembly into fibrils have been associated with chronic inflammatory conditions and certain neoplasms, which lead to the deposition of AA amyloid in organs such as the liver, spleen, and kidney (systemic amyloidosis). For this reason, SAA is a highly significant molecular target. Insight into the structural biology of the promoter element that determines the combinatorial control of gene expression by different transcriptional factors is essential for understanding the dysfunction of SAA gene expression. The highly GC‐rich region of the human SAA promoter is crucial for basal promoter activity and likely to assume a DNA secondary structure, such as G‐quadruplex (GQ). GQs often serve as transcriptional silencer elements. Stabilization of GQ is possible with small molecule therapeutics to modulate gene expression. In the present study, G‐quadruplex formation has been isolated from the GC‐rich region of the 5’‐untranslated region (5’UTR) of SAA2. An electrophoretic mobility shift and thioflavin T fluorescence assays were used to confirm the formation of a biologically relevant intramolecular structure. The structure of the G‐quadruplex is currently under investigation by circular dichroism and DMS footprinting. These data characterizing the formation of a unique secondary structure in SAA gene are promising to develop novel anti‐amyloid therapy for the treatment of systemic amyloidosis.
Alpha‐synuclein is an abundant neuronal protein composed of 140 amino acids. Compelling evidence suggests that alpha‐synuclein can acquire a neurotoxic feature that kills dopamine secreting nerve cells in horses afflicted with Cushing's disease (known as pituitary pars intermedia dysfunction, PPID). Using bioinformatic analysis, we have identified that equine alpha‐synuclein has a high propensity to convert (misfold and aggregate) into this toxic form. While alpha‐synuclein is thought to play a pathogenic role in horses with equine PPID, it is unclear how alpha‐synuclein aggregates. We hypothesize that specific region(s) and/or amino acid variation(s) in equine alpha‐synuclein contribute to its misfolding. To test this hypothesis, we designed a bank of fragmented peptides to identify the region(s) most prone to aggregation. Bioinformatic analysis of equine alpha‐synuclein with Tango program indicated a similar propensity to aggregate as human alpha‐synuclein (Fig. 1). Region 62‐87 of the equine alpha‐synuclein peptide is the region most prone to aggregation according to Thioflavin T fluorescence assay. This fragment peptide 62‐87 is capable of generating mature fibrils as validated by electron microscopy. The discovery of the region responsible for equine alpha‐synuclein misfolding is relevant for the development of pharmacological agents that block the formation of alpha‐synuclein toxic aggregates.
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