microRNA profiling of human PSP, CBD, and DLBD brain tissues compared to normal controls

Heather Estrella1, Randy Soriano1, Kimberlee Fischer1, Jeff Friedman2, Dennis Dickson3, Adam Pavlicek1

, 1Regulus Therapeutics, San Diego, CA, 2CurePSP, San Diego, CA, 3Mayo Clinic, Jacksonville, FL

Abstract ResultsProgressive 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

FD

R

miR

Down-regulated miRPSPCBDNormal

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

FD

R

miR

PSP CBD DLBD Normal

FC

FD

R

miR

PSP CBD DLBD Normal

FC

FD

R

miR