microRNA profiling of human PSP, CBD, and DLBD brain tissues compared to normal controls Heather Estrella 1 , Randy Soriano 1 , Kimberlee Fischer 1 , Jeff Friedman 2 , Dennis Dickson 3 , Adam Pavlicek 1 , 1 Regulus Therapeutics, San Diego, CA, 2 CurePSP, San Diego, CA, 3 Mayo Clinic, Jacksonville, FL Abstract Results Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD) are sporadic parkinsonian disorders with Tau pathology that share many molecular and clinical features. Patients with these disorders experience relentless neurodegeneration and typically succumb within 5 years of diagnosis. There are currently no disease modifying therapies for PSP or CBD, representing a significant unmet medical need. In this study, we performed comprehensive microRNA profiling in brain samples from PSP (n=10) and CBD (n=10) independent human patients. As control groups, we used brain samples from patients with no neurodegenerative disease (n=10). We also included samples from patients with Diffuse Lewy Body Disease (DLBD) that lack Tau involvement (n=10). From each subject, the frontal cortex and cerebellum were isolated (total 80 samples) and profiled for microRNA expression using the Human NanoString microRNA assays version 2. Data were normalized by positive controls and total microRNA signal, background adjusted, and batch adjusted using ComBat (R library sva). microRNAs with log2 expression > 2 in less than 10% of samples were removed from analysis (low expression). 461 sufficiently expressed microRNAs passed this cut-off and were used in subsequent differential expression analyses. Many microRNAs were differentially expressed in PSP, CBD, and DLBD patients compared to controls. Expression changes in the frontal cortex were much more pronounced compared to the cerebellum. In the cortex, we found 53 microRNAs commonly deregulated at a false discovery rate (FDR) < 20% in both PSP and CBD samples compared to normal controls. Most of the significant microRNAs, including many brain-specific transcripts, were downregulated indicating potential cell loss. A similar pattern of downregulation was observed in the cortex of DLBD samples compared to normal controls. Many of the differentially expressed microRNAs identified in this study have been implicated in pathogenesis of other neurodegenerative diseases and may represent common drivers of neurodegenerative disorders warranting further investigations. Results Society for Neuroscience, November 12-16, 2016, San Diego, CA Poster #: 514.14-Z10 Abbreviations • PSP Progressive Supranuclear Palsy • CBD Corticobasal Degeneration • DLBD Diffuse Lewy Body Disease • FC Fold-change (log2) • PV or P.Value Student’s T -test p-value • FDR False-discovery rate • Many significant microRNAs detected • Mostly loss of microRNA expression in DLBD compared to normal Figure 4. DLBD vs. normal: frontal cortex Methods Study Design Each disease had 10 subjects with both frontal lobe and cerebellum with the exception of the normal group which had 9 (1 sample failed quality control) Profiling • Samples were run on 7 NanoString human microRNA cartridges (1 cartridge for 12 samples; NS_H_miR_v2) • Samples were randomized with respect to tissue types and disease status • Data normalized by positive controls and total microRNA signal, background adjustment • Batch adjustment using ComBat (R library sva) Analysis • microRNAs with log2 expression > 2 in less than 10% of samples were removed from analysis (low expression; 461 microRNA assays passed this cut-off) • Differential gene expression analysis based on moderated t-test (R limma package) • P-values corrected by Benjamini-Hochberg false discovery rate (FDR) • FDR cutoff set to 0.2 (20%) for volcano plots and Venn diagrams Objectives To determine the overall microRNA regulation for PSP, CBD and DLBD vs. normal frontal lobe or cerebellum and common microRNA differentiation amongst these neurodegenerative diseases • More dramatic microRNA expression changes found in the frontal cortex compared to cerebellum • There appears to be a loss of microRNAs in PSP and CBD compared to normal brain tissues • Several commonly up-regulated microRNAs identified in PSP and CBD samples may represent common drivers of neurodegenerative disorders Conclusions Figure 2. CBD vs. normal: frontal cortex • Many significant microRNAs detected • Mostly loss of microRNA expression in CBD compared to normal • Many differentially expressed microRNA • Mostly loss of microRNA expression in PSP compared to normal Figure 1. PSP vs. normal: frontal cortex • Fewer differentially expressed microRNAs found compared to frontal cortex • Similar results for CBD and DLBD (not shown) Figure 3. PSP vs. normal: cerebellum Overlap significant miR (FDR < 0.2) PSP CBD DLBD Normal FC FDR miR Down-regulated miR PSP CBD Normal Up-regulated miR Figure 5. Overlap between PSP vs. normal & CBD vs. normal frontal lobe differential expression sets • Differential analysis including both PSP and CBD vs. normal, adjusted for disease type • Many consistently deregulated microRNAs found at FDR < 20% • 14 consistently up-regulated PSP CBD DLBD Normal FC FDR miR PSP CBD DLBD Normal FC FDR miR PSP CBD DLBD Normal FC FDR miR