Gene-Expression Profiling Suggests Impaired Signaling via the Interferon Pathway in Cstb-/- Microglia.

Inken Körber,Elisabet Einarsdottir,Juha Kere,Anna-Elina Lehesjoki,Tarja Joensuu,Kaarel Krjutškov,Shintaro Katayama,Paula Hakala

doi:10.1371/journal.pone.0158195

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb-/- mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb-/- mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb-/- microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb-/- microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune- and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.

Highlights

The neurodegenerative disease progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive disease with onset between 6 and 16 years of age
To pinpoint the relevant expression changes in Cstb-/- microglia, we focused on the transcript clusters with an absolute fold change (FC) of at least 1.3 and a corrected p < 0.05, and detected 155 differentially expressed genes (DEGs; S1 Table)
Our previous findings of an early activation and dysfunction of Cstb-/- microglia with consequent neuroinflammation [14, 19] and an upregulation of genes linked to immune system processes in cerebella of one-month-old Cstb-/- mice [18] strongly supported a contribution of microglial dysfunction and inflammatory processes in the pathogenesis of EPM1

Summary

Introduction

The neurodegenerative disease progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive disease with onset between 6 and 16 years of age. It is characterized by progressive, stimulus-sensitive, and action-activated myoclonus, which is resistant to medication and severely impairs patients’ everyday life [1]. Loss-of-function mutations in the cystatin B (CSTB) gene that encodes the cysteine protease inhibitor CSTB [2] underlie EPM1. The increased proteolytic activity of cathepsin B identified in CSTB-deficient cerebellar granule neurons and of cathepsin B, L, and S in EPM1 patient lymphoblastoid cells implies that CSTB functions as a cathepsin inhibitior in vivo [6, 7]. The underlying molecular mechanisms and how the loss of CSTB causes the phenotype of EPM1 remain unknown

Methods

Results

Discussion

Conclusion