Gene Expression Alterations in the Cerebellum and Granule Neurons of Cstb−/− Mouse Are Associated with Early Synaptic Changes and Inflammation

Tarja Joensuu,Mikael Segerstråle,Paula Hakala,Esa R Korpi,Saara Tegelberg,Outi Kopra,Heidi Pehkonen,Iiris Hovatta,Jaana Tyynelä,Eva Reinmaa,Tomi Taira,Anna-Elina Lehesjoki

doi:10.1371/journal.pone.0089321

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited neurodegenerative disease, manifesting with myoclonus, seizures and ataxia, caused by mutations in the cystatin B (CSTB) gene. With the aim of understanding the molecular basis of pathogenetic events in EPM1 we characterized gene expression changes in the cerebella of pre-symptomatic postnatal day 7 (P7) and symptomatic P30 cystatin B -deficient (Cstb−/−) mice, a model for the disease, and in cultured Cstb−/− cerebellar granule cells using a pathway-based approach. Differentially expressed genes in P7 cerebella were connected to synaptic function and plasticity, and in cultured cerebellar granule cells, to cell cycle, cytoskeleton, and intracellular transport. In particular, the gene expression data pinpointed alterations in GABAergic pathway. Electrophysiological recordings from Cstb−/− cerebellar Purkinje cells revealed a shift of the balance towards decreased inhibition, yet the amount of inhibitory interneurons was not declined in young animals. Instead, we found diminished number of GABAergic terminals and reduced ligand binding to GABAA receptors in Cstb−/− cerebellum. These results suggest that alterations in GABAergic signaling could result in reduced inhibition in Cstb−/− cerebellum leading to the hyperexcitable phenotype of Cstb−/− mice. At P30, the microarray data revealed a marked upregulation of immune and defense response genes, compatible with the previously reported early glial activation that precedes neuronal degeneration. This further implies the role of early-onset neuroinflammation in the pathogenesis of EPM1.

Highlights

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM 254800) is an autosomal recessively inherited neurodegenerative disease characterized by stimulus-sensitive myoclonus, tonic-clonic seizures and ataxia with the disease onset at 6–16 years of age [1]
This study is based on the hypothesis that the pathogenetic mechanisms of EPM1 are reflected in altered gene expression patterns
To identify molecular defects associated with cystatin B gene (CSTB) deficiency, we used cerebellum as a model which shows striking pathology in Cstb2/2 mice [4,6], and is affected in human EPM1 patients [21]

Summary

Introduction

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM 254800) is an autosomal recessively inherited neurodegenerative disease characterized by stimulus-sensitive myoclonus, tonic-clonic seizures and ataxia with the disease onset at 6–16 years of age [1]. EPM1 is caused by loss-of-function mutations in the cystatin B gene (CSTB), the most common of which is an expansion of a dodecamer repeat in the promoter region of the gene [2,3]. A mouse model for the disease, the Cstbdeficient (Cstb2/2) mouse, presents with many of the clinical features of EPM1, especially myoclonus starting at the age of 1 month and progressive ataxia manifesting around 6 months of age [4]. One of the major neuropathological phenotypes in Cstb2/2 mice is a severe loss of cerebellar granule neurons due to apoptotic death. We recently reported striking, early microglial activation in Cstb2/2 brain, which precedes the emergence of myoclonus and is followed by widespread astroglial activation and selective neuronal loss [6]

Methods

Results

Conclusion